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c./ 



UILU-WRC-78-0133 

RESEARCH REPORT NO. 133 



Rapid Assessment of Water Quality, 
Using the Fingernail Clam, 
tAuscuWum transversum 



UJ3C 



By Kevin B. Anderson 
and Richard E. Sparks 



ILLINOIS NATURAL HISTORY SURVEY 
URBANA, ILLINOIS 



UNIVERSITY OF ILLINOIS 
ATURBANACHAMPAIGN 
WATER RESOURCES 
CENTER 



Anthony A. Paparo 

SCHOOL OF MEDICINE AND DEPARTMENT OF ZOOLOGY 
SOUTHERN ILLINOIS UNIVERSITY, CARBONDALE 



APRIL 1978 



OofieGonn> copy 

(^ . If, 39, ud) 



WRC RESEARCH REPORT NO. 133 



RAPID ASSESSMENT OF WATER QUALITY, 
USING THE FINGERNAIL CLAM, MUSCULIUM TRANSVERSUM 



by 



Kevin B. Anderson* and Richard E. Sparks* 

ILLINOIS NATURAL HISTORY SURVEY 

RIVER RESEARCH LABORATORY 

Havana, Illinois 62644 

and 

Anthony A. Paparo* 

SCHOOL OF MEDICINE AND DEPARTMENT OF ZOOLOGY 

SOUTHERN ILLINOIS UNIVERSITY 

Carbondale, Illinois 62901 

*co-authors 



FINAL REPORT 
Project No. B-097-ILL 



This project was partially supported by the U.S. 
Department of the Interior in accordance with 
the Water Resources Research Act of 1965, P.L. 
88-379, Agreement No. USDI 14-31-0001-6072 



UNIVERSITY OF ILLINOIS 
WATER RESOURCES CENTER 
2535 Hydrosystems Laboratory 
Urbana, Illinois 61801 



April, 1978 



Digitized by the Internet Archive 

in 2010 with funding from 

CARLI: Consortium of Academic and Research Libraries in Illinois 



http://www.archive.org/details/rapidassessmentoOOande 



NOTE 



The original title of this research project was "Rapid Assessment of 
Water Quality Using the Fingernail Clam, Sphaerium transversum ". The 
scientific name of the clam was changed to Musculium transversum while 
the research was in progress. Some of the figures in this report use the 
older scientific name, Sphaerium transversum . 



iii 



TABLE OF CONTENTS 

Page 

ABSTRACT 1 

INTRODUCTION AND BACKGROUND 2 

METHODS 8 

General Approach 8 

Collection of Fingernail Clams 9 

Rapid Screening Methods 9 

Acute Bioassay Methods 16 

Chronic Bioassay Methods 20 

Elemental Analysis of Shells 23 

RESULTS AND DISCUSSION 24 

Water Quality in the Illinois River in the 1950' s 24 

Comparison of Water Quality in the Mississippi and Illinois 

Rivers in 1975 25 

Reliability of the Rapid Screening Methods 30 

The Gaping Response as an Indicator of Death 31 

Acute and Chronic Bioassay Methods with Juvenile and Adult 

Fingernail Clams 31 

Response of Clams to Light and Darkness 33 

Response of Clams to Temperature 35 

Response of Clams to Dissolved Oxygen 37 

Response of Clams to Sodium Nitrate and Sodium Sulfate 40 

Response of Clams to Sodium Cyanide and Potassium Cyanide 43 

Response of Clams to Lead Nitrate, Copper Sulfate, and Zinc 

Sulfate 43 



Table of Contents (continued) 

Page 

Response of Clams to Potassium Chloride 46 

Response of Gill Preparations 46 

Acute Response of Intact Clams to Potassium Chloride 50 

Chronic Response of Intact Fingernail Clams to Potassium 

Chloride 53 

Effect of Water Hardness in Modifying Toxicity of 

Potassium Chloride 58 

Effect of Temperature in Modifying Toxicity of Potassium 

Chloride 59 

Response of Clams to Ammonium Chloride 61 

Response of Gill Preparations 61 

Effect of Oxygen in Modifying Toxicity of Ammonium 

Chloride 63 

Chronic Response of Intact Clams to Ammonium Chloride 65 

Effect of Sublethal Exposure to Ammonium Chloride on 

Subsequent Response to Stress 69 

Relative Sensitivity of Fingernail Clams and Other 

Aquatic Organisms to Ammonia 74 

Relationship between Ammonia Levels in the Illinois and 

Mississippi Rivers and Ammonia Levels Which Affected 

Fingernail Clams in Laboratory Experiments 75 

Response of Clams to Suspended Particles 81 

Response of Clams to Raw Illinois River Water 84 

Response of Gill Preparations 84 

Chronic Response of Clams to Raw Illinois River Water 86 

Analysis of Shells from Deformed Clams 87 

SUMMARY 89 

RECOMMENDATIONS 97 

RELATION OF THIS RESEARCH TO WATER RESOURCES PROBLEMS 98 



vi 



Table of Contents (continued) 



Page 



ACKNOWLEDGEMENTS 99 

LITERATURE CITED 100 

APPENDIX A ~ TEST CONDITIONS AND RESULTS OF ACUTE AND CHRONIC 

BIOASSAYS, TABLES 7-26 104 

APPENDIX B — PUBLICATIONS AND THESIS RESULTING FROM THIS 

RESEARCH 114 

Publications 114 

Thesis 115 



LIST OF FIGURES 



Figure 1. Live fingernail clams, Musculium transversum , which 
were raised in the laboratory. 

Figure 2. Use of the Illinois River by lesser scaup ducks 

plummeted following the die-off of fingernail clams 
in 1955 and has never recovered. 



Figure 3. Live fingernail clams, Musculium transversum , were 
obtained from the Keokuk Pool, Mississippi River, 
using a boat specially equipped with a crane and a 
Ponar grab sampler. 10 

Figure 4. Apparatus for measuring the effects of water quality 

factors on the ciliary beating rate of clam gills. 12 

Figure 5. Generalized clam anatomy, showing the visceral 

ganglion and the gill, which are used in the cilia 
monitoring apparatus. 14 

Figure 6. Scanning electron micrograph of gill filaments from 
the blue mussel, Mytilus edulis , showing the gill 
filaments, lateral cilia, latero-frontal cilia, and 
frontal cilia, which are similar to those in the 
fingernail clam. 15 

Figure 7. Toxicant diluters which delivered test solutions or 
well water to aquaria which served as test chambers 
for the chronic bioassays. 21 

Figure 8. Light inhibits the beating of lateral cilia on the 

gills of two Sphaeriacean clams ( Musculium transversum 
and Corbicula manilensis ) and the unrelated intertidal 
mussel (Mytilus edulis ) . 34 

Figure 9. Ciliary beating response of gills from large and small 
fingernail clams ( Musculium transversum ) , the Asiatic 
clam ( Corbicula manilensis ) and the blue mussel 
( Mytilus edulis) to temperature. 36 

Figure 10. Beating rate of cilia on gills from large fingernail 
clams is proportional to the increase in water 
temperature. 38 

Figure 11. Beating rate of cilia on gills from small clams is 

proportional to the increase in water temperature. 38 



List of Figures (continued) 



Page 



Figure 12. The ciliary beating rate of gills from large and 
small fingernail clams increased as the oxygen 
concentration of the water increased. 



39 



Figure 13. The ciliary beating rate of gills from large and 

small fingernail clams ( Musculium transversum ) , Asiatic 
clams ( Corbicula manilensis ) , and the blue mussel 
( Mytilus edulis ) declined when the oxygen level was 
reduced from 10 to 2 mg/1, and recovered when the 
oxygen level was restored to 10 mg/1 40 

Figure 14. Ciliary beating response of gills from small clams. 

A potassium concentration of 39 mg/1 maintains a basal 
rate; higher concentrations are cilio-inhibitory . 46 

Figure 15. Ciliary beating response of gills from small clams. 

A potassium concentration of 39 mg/1 maintains a basal 
rate, while lower concentrations (3.9 and 0.039 mg/1) 
temporarily excite the cilia. 47 



Figure 16. Ciliary beating response of gills from small clams. 

Continuous exposure of the gills to potassium concen- 
trations of 0.039 and 0.0039 mg/1 causes an increase 
in ciliary beating rate for four days, followed by a 
return to nearly the basal rate or slightly above. 



47 



Figure 17. Ciliary beating response of gills from large clams. 
A potassium concentration of 0.039 mg/1 maintains a 
basal rate; higher concentrations are cilio-irihibitory . 48 



Figure 18. Ciliary beating response of gills from large clams. 
Potassium concentrations of 0.0039 and 0.00039 mg/1 
excite the cilia; while 0.000039 and 0.0000039 fail 
to maintain the basal rate. 



48 



Figure 19. Ciliary beating rate of gills from large clams fails 
to recover from inhibiting effect of potassium 
withdrawal. 



49 



Figure 20. Gills from large clams show a greater lag than gills 
from small clams in response to a cilio-excitatory 
level of potassium. 



50 



Figure 21. Comparison of acute toxicity curves, adult test Al 

and juvenile test Jl. 51 

Figure 22. Comparison of acute toxicity curves of adult test A2 

and juvenile test J2. 52 



List of Figures (continued) 



Page 



Figure 23. In the first chronic bioassay with potassium (Kl), 

there were no significant differences in the mortality 
of clams exposed to the different potassium 
concentrations. 53 



Figure 24. In the first chronic bioassay with potassium (Kl) , 
there were no significant differences in the growth 
(in length) of clams exposed to the different 
potassium concentrations. 



54 



Figure 25. In the second chronic bioassay with potassium (K2) 
the mortality of clams exposed to 275 mg/1 was sig- 
nificantly greater than the mortality in the lower 
concentrations and in water with no added potassium. 



55 



Figure 26. In the second chronic bioassay with potassium (K2) , 
there were no significant reductions in growth of the 
clams exposed to the different potassium concentrations. 56 



Figure 27. Clams exposed to potassium in hard water (314 mg/1 

as CaCO-) did not die as rapidly as clams exposed to 
potassium in softer water (243 mg/1 as CaCO„) . 

Figure 28. Juvenile fingernail clams died more rapidly in warm 
water than in cool water when exposed to potassium 
chloride, but the lethal thresholds at the two tem- 
peratures were probably almost the same. 



59 



60 



Figure 29. Ciliary beating response of gills from large and 
small fingernail clams to un-ionized ammonia. 

Figure 30. Ciliary beating response of gills from the Asiatic 
clam ( Corbicula manilensis ) , a freshwater mussel 
(Elliptio complanata ) , and a marine mussel ( Mytilus 
edulis) , to un-ionized ammonia. 



61 



62 



Figure 31. Ciliary beating rates of gills from small clams did 
not decrease when the gills were exposed to .07 mg/1 
NH„-N and oxygen concentrations of 14 mg/1 or more 
(150% or more of the oxygen saturation concentration 
of 9.18 mg/1). 



64 



Figure 32. The ciliary beating rates of gills from large finger- 
nail clams decreased markedly when the gills were 
exposed to .07 mg/1 NH„-N, and oxygen concentrations 
below 12 mg/1 (130% of saturation). 



64 



Figure 33. Un-ionized ammonia concentrations of .93 and .59 mg/1 
(as NH„-N) caused significant mortality among 
fingernail clams after 42 days of exposure. 



65 



List of Figures (continued) 



Page 



Figure 34. Results of chronic bioassay mi^l. 

Figure 35. An un-ionized ammonia concentration of 1.20 mg/1 

caused significant mortality among fingernail clams 
after 14 days of exposure, and a concentration of 
0.60 mg/1 caused significant mortality after 42 days, 

Figure 36. Un-ionized ammonia concentrations of 0.34 and 0.60 
mg/1 (NH -N) significantly reduced the growth of 
fingernail clams after 14 days of exposure. 



66 



67 



68 



Figure 37. The ciliary beating response to potassium of gills 
from clams previously exposed for 44 days to sub- 
lethal NH„-N concentrations of 0.1 and 0.2 mg/1 was 
markedly altered in comparison to the response of gills 
from clams not previously exposed to ammonia. 70 

Figure 38. When clams are exposed to sublethal concentrations of 
un-ionized ammonia (0.1 and 0.2 mg/1 NH„-N) for 44 
days, their gills are sensitized to subsequent addi- 
tions of ammonia. 71 



Figure 39. Previous exposure of clams to sublethal concentrations 
of un-ionized ammonia reduces the maximum ciliary 
response of their gills to temperature and also 
reduces the temperature range in which normal ciliary 
activity can be maintained. 

Figure 40. Previous exposure of fingernail clams to sublethal 
concentrations of un-ionized ammonia reduces the 
ability of the gills to increase their ciliary beating 
rate in response to increasing concentrations of 
oxygen. 

Figure 41. Low oxygen concentrations cause greater inhibition 
of ciliary beating rates on gills from clams 
previously exposed to sublethal concentrations of 
ammonia than on gills from clams not exposed to 
ammonia. 



72 



73 



74 



Figure 42. The peak number of fingernail clams in bottom 

samples from Keokuk Pool, Mississippi River declined 
by 90% in 1976-1977. 



77 



Figure 43, 



The growth of fingernail clams in the Keokuk Pool, 
Mississippi River, was reduced in 1976. 



78 



xii 



List of Figures (continued) 

Page 

Figure 44. Mississippi River discharge at Keokuk, Iowa, showing 

the effects of the 1976-1977 drought. 79 

Figure 45. The concentration of un-ionized ammonia (NH„-N) in 
the Keokuk Pool, Mississippi River, was greater in 
1976 than in 1973. 80 

Figure 46. The ciliary activity of gills from small fingernail 
clams was almost completely inhibited when the gills 
were exposed to water taken from the Illinois River. 85 

Figure 47. The white arrows show the curved shells which 

developed when the clams were exposed to Illinois 

River water which had been contaminated with metals. 86 



LIST OF TABLES 



Page 



Table 1. Source and Chemical Characteristics of the Test Waters 

and Test Light-Dark Cycles 17 

Table 2. Mean Values of Water Quality Factors Which Occurred at 

Higher Levels in the Middle Section of the Illinois * 
River, Where Fingernail Clams Died Out, Than in the 
Keokuk Pool, Mississippi River, Where Fingernail Clams 
Were Still Abundant Through 1975 27 

Table 3. Median Values of Water Quality Factors Which Occurred 
at Higher Levels in the Middle Section of the Illinois 
River, Where Fingernail Clams Died Out, Than in the 
Keokuk Pool, Mississippi River, Where Fingernail Clams 
Were Still Abundant Through 1975 28 

Table 4. Maximum Values of Water Quality Factors Which Occurred 
at Higher Levels in the Middle Section of the Illinois 
River, Where Fingernail Clams Died Out, Than in the 
Keokuk Pool, Mississippi River, Where Fingernail Clams 
Were Still Abundant Through 1975 29 

Table 5. Effects of Sodium Nitrate, Sodium Sulfate, Lead Nitrate, 
Copper Sulfate, and Zinc Sulfate on the Average Ciliary 
Beating Rate of the Gills of Musculium transversum 42 

Table 6. Effect of Oxygen Tension and Suspended Particles on the 
Average Rate of Transport of Particles on the Gills of 
Musculium transversum 82 

APPENDIX A 

Table 7. Test Conditions for Adult Test Al 104 

Table 8. Results of Test Al 104 

Table 9. Test Conditions for Adult Test A2 105 

Table 10. Results of Test A2 105 

Table 11. Test Conditions for Juvenile Test Jl 106 

Table 12. Results of Test Jl 106 

Table 13. Test Conditions for Juvenile Test J2 107 

Table 14. Results of Test J2 107 



List of Tables (continued) 

Page 

Table 15. Test Conditions for Juvenile Test J3 108 

Table 16. Results of Test J3 108 

Table 17. Test Conditions for Juvenile Test J4 109 

Table 18. Results of Test J4 109 

Table 19. Test Conditions for Chronic Bioassay Kl 110 

Table 20. Results of Chronic Bioassay Kl 110 

Table 21. Test Conditions for Chronic Bioassay K2 111 

Table 22. Results of Chronic Bioassay K2 111 

Table 23. Test Conditions for Chronic Bioassay NH 2 112 

Table 24. Results of Chronic Bioassay NH 2 112 

Table 25. Test Conditions for Chronic Bioassay KE^S 113 

Table 26. Results of Chronic Bioassay NH„3 113 



xvi 



ABSTRACT 



Apparatus and methods were developed for testing the effects of water 
quality factors on the ciliary beating rate of clam gills. Musculium trans- 
versum was chosen as the test organism because it is a major food source for 
fish and waterfowl, and because it has died out in areas of the Illinois River 
where it was formerly abundant. Populations of this fingernail clam also de- 
clined recently in the Keokuk Pool, Mississippi River, an important feeding 
area for migratory waterfowl and commercially valuable species of fish. 

The ciliary beating response is extremely sensitive. For example, a zinc 
concentration of .00006 yg/1 produced a statistically significant reduction in 
the ciliary beating rate of gills from large fingernail clams. Gills from small 
clams were much less sensitive, requiring .06-. 6 yg/1 zinc to produce the same 
response. Concentrations of potassium and un-ionized ammonia which inhibited 
the ciliary beating response of gills from small clams were quite close to the 
concentrations which reduced the survival or growth of intact clams during 
chronic bioassays. The threshold concentration of potassium for cilia inhibi- 
tion of small clams lay between 39 and 390 mg/1. The maximum acceptable toxi- 
cant concentration (MATC) for long-term survival of fingernail clams lay between 
195 and 275 mg/1 potassium. Un-ionized ammonia concentrations of .08-. 09 mg/1 
inhibited the cilia of small clams, and the growth of the clams was reduced at 
concentrations between .20 and .34 mg/1 NH„-N. 

In addition to potassium and ammonia, the following factors were tested 
singly or in combination: light, temperature, dissolved oxygen, sodium nitrate, 
sodium sulfate, cyanide, lead, copper, zinc, suspensions of silica particles, 
suspensions of illite clay particles, and raw Illinois River water. Comparison 
of the levels of these water quality factors in the Illinois and Mississippi 
Rivers with the levels which had detrimental effects on the clam gills sug- 
gests that un-ionized ammonia and heavy metals may have affected fingernail 
clams in the Illinois River in the 1950 's and in the Mississippi River in 
1976-1977. These tentative conclusions should be validated using chronic 
bioassays in which fingernail clams are exposed to conditions simulating those 
in the Mississippi River in 1976-1977, and by deletion bioassays in which cer- 
tain components are removed from raw Illinois River water and the survival, 
growth, and reproduction of clams in the treated water are measured. 

KEY WORDS: water pollution effects, bioassay, bioindicators , animal 
physiology, fingernail clams, Sphaerium transversum , Musculium transversum , 
Sphaeriidae, heavy metals, silt, heat, dissolved oxygen, cyanide, ammonia, 
potassium, suspended solids, suspended sediment, sodium nitrate, sodium 
sulfate, lead, copper, zinc, Keokuk Pool, Mississippi River, Illinois River, 
Asiatic clam, Corbicula manilensis , blue mussel, Mytilus edulis , Elliptio 
complanata . 



INTRODUCTION AND BACKGROUND 

Fingernail clams (Family Sphaeriidae) are dominant bottom-dwelling 
animals in some waters of the midwestem part of the United States. They 
are found in major rivers (Gale, 1969: v) , in lakes (Emmling, 1974: 11), 
even in bottomlands which are only intermittently flooded (Hubert, 1972: 
177-178), and are key links in food chains leading from nutrients in water 
and mud to fish and ducks which are utilized by man. Fingernail clams 
filter algae, bacteria, and organic matter from water. Because the clams are 
small (less than 15 mm long when full-grown) , they in turn are readily con- 
sumed by ducks and bottom-feeding fish. A short food chain of this type 
can support a larger biomass at the top level (ducks and fish) than a 
longer one. 

The fingernail clam, Musculium transversum (Say, 1829), shown in 
Figure 1, prefers big river habitat with a silt bottom where peak numbers 
may exceed 100,000/m as in the Keokuk Pool, Mississippi River (Gale, 1969: 
v) . Keokuk Pool is a 74-km (46-mile) section of the mainstem of the Missis- 
sippi extending from Lock and Dam 19 at Keokuk, Iowa to Lock and Dam 18 
near Burlington, Iowa. Ranthum (1969) and Jude (1968, 1973) studied the food 
habits of fish in the Keokuk Pool and found that at certain times of the 
year fingernail clams made up 100% by volume of the diets of carp ( Cyprinus 
carpio) and smallmouth buffalo ( Ictiobus bubalus ) , and 10 to 70% of the diets 
of black bullhead ( Ictalurus melas ) , gizzard shad ( Dorosoma cepedianum ) , 
pumpkinseed ( Lepomis gibbosus ) , bigmouth buffalo ( Ictiobus cyprinellus ) , 
freshwater drum ( Aplodinotus grunniens ) , and bluegill (Lepomis macrochirus ) . 
These fishes include commercial, sport, and forage species. 




Figure 1. Live fingernail clams, Musculium transversum , which were raised 
in the laboratory. The large individuals are approximately 
8 ram across the longest dimension of the shell, and will grow 
to 15 mm. The small, light-colored clam in the upper left is 
new-born and is approximately 2 mm across. The clam in the 
center has extended its siphons, and the individual just to 
the right and below center has extended its foot. 



Thompson (1973: 379) estimated that lesser scaup ducks ( Aythya af finis ) , 
ring-necked ducks ( Aythya collaris ) , canvasbacks ( Aythya valisineria ) , common 
goldeneyes ( Bucephala clangula ), and ruddy ducks ( Oxyura jamaicensis ) consumed 
2.2 million kg (4.8 million pounds) of fingernail clams in Keokuk Pool during 
the fall migration of 1967, or approximately 24% of the standing crop of 
fingernail clams at the Keokuk Pool (Gale, 1973: 175). Thompson found that 
the clams made up 85-95% by volume of food items taken by these ducks in the 
spring of 1967. The Keokuk Pool attracts about 20 million diving duck-days 
of use per year (Personal communication, F.C. Bellrose, Wildlife Specialist, 
Illinois Natural History Survey, August, 1976) and has been characterized as 
the most important inland body of water for diving ducks in North America 
(Trauger and Serie, 1974: 71). 

In the mid-1950 's fingernail clams virtually disappeared from a 100-mile 
section of the Illinois River, a tributary of the Mississippi River, due to 
unknown causes (Paloumpis and Starrett, 1960: 406-435; Anderson, 1977: 3, 48- 
54). A survey of the bottom fauna of the Illinois River by Anderson in 1975 
(Anderson, 1977: 5) revealed that fingernail clams were still absent from the 
middle reach of the River, where they had been abundant prior to the die-off 
in the 1950 's. Fingernail clams can quickly repopulate an area when conditions 
are suitable. The clams can complete a life cycle in 33 days (Gale, 1969: v) , 
and remnant "seed" populations are available in tributary streams and in an 
area of Peoria Lake where spring flow occurs (Anderson, 1977: 48-54). Some 
factor or factors in the Illinois River currently prevent the clams from 
recolonizing areas where they were formerly abundant. The unknown factor is 
probably the same one which caused the original die-off in the 1950' s, 
although it is possible that the factor which eliminated the clams has been 
replaced by another factor which prevents recolonization. 



As a result of the die-off of fingernail clams, the number of diving 
ducks, such as lesser scaups and canvasbacks , using the Illinois River during 
migration declined drastically (Mills, Starrett, and Bellrose, 1966: 18-20) 
and has never recovered since that time (Figure 2) . Some of the ducks evi- 
dently shifted their migration route to the Mississippi River, where finger- 
nail clams were still available (Mills et al. , 1966: 18). Carp in the section 
of the Illinois River where the die-off of fingernail clams occurred are 
measurably thinner and smaller than downstream fish, probably because of 
poorer nutrition (Mills et al. , 1966: 17). In the 1960's, fingernail clams 
formed 50.2 percent by volume of the food items taken by carp in sections of 
the river unaffected by the die-off, but only one clam was found in a carp 
from the affected section (Starrett, 1972: 151). 

The disappearance of the fingernail clams in the Illinois River and the 
dramatic ecological repercussions of that disappearance illustrate the 
need for assessing water quality effects on lower organisms, in addition to 
fish. Apparently fingernail clams are more sensitive than fish to some factor 
in Illinois River water. If the effects of water quality factors on clams 
can be determined, it might be possible to make conditions in the Illinois 
River suitable for fingernail clams again and to prevent a similar ecological 
disaster from occurring in the Mississippi River. Restoration of fingernail 
clam populations in the Illinois River would dramatically increase fish pro- 
duction and diving duck utilization of the river. 

In order to determine the effects of water quality on fingernail clams, 
a type of organism for which no standard testing methods exist, three methods 
were developed during this project: a rapid screening method, an acute bio- 
assay method, and a chronic bioassay method. Shells of clams which had been 



Figure 2. Use of the Illinois River by lesser scaup ducks plunnneted 
following the die-off of fingernail clams in 1955 and 
has never recovered. 



2.520 H 



^ 2.160- 



§ L800 



m^o 



1.080 



m 720- 



P 360- 



LESSER SCAUP 

I 1 = MISSISSIPPI RIVER VALLEY 
ILLINOIS RIVER VALLEY 




I I I I I i I 

1946 48 50 52 54 56 58 60 62 64 66 68 70 72 74 75 



YEAR 



exposed to Illinois River water were also subjected to elemental analysis 
using X-ray microprobe techniques. The development of rapid screening methods 
is of particular importance, because of the length of time required to 
complete most acute and chronic bioassays and because of the accelerating 
rate of production of new chemicals which should be tested before being re- 
leased to the environment. 



METHODS 



General Approach 

It would take one lifetime, or perhaps several, to test all possible 
factors which might affect survival of fingernail clams. We took two approaches 
to reduce the burden of testing to a manageable size. Our first approach 
was to compare water quality in the Illinois River before and after the die- 
off of fingernail clams, and to compare recent water quality in the Illinois 
River with water quality in the Keokuk Pool, Mississippi River, where finger- 
nail clams are still abundant. We used the annual summaries of data from 
the Water Quality Sampling Program of the Illinois Environmental Protection 
Agency (lEPA) . The lEPA was established in 1970. Older data were obtained 
from the Illinois State Water Survey, which has had a water quality sampling 
station on the Illinois River at Peoria. We also used some additional water 
quality data obtained during biological surveys conducted by the Illinois 
Natural History Survey. 

The second approach was to develop a method for rapidly assessing the 
effects of water quality factors on fingernail clams. The rapid method measures 
the average rate of beating of lateral cilia on excised clam gills. The beating 
rate of the lateral cilia is precisely regulated and coordinated. Changes 
in beating rate, or other changes, such as stoppage of the lateral cilia or 
a change from a metachronal to a synchronal pattern, occur rapidly (within 
15 min to 1 h) in response to a variety to stimuli, and can be observed and 
measured within minutes by the method to be described in more detail below. 
Since the lateral cilia on the gills of the clam produce the water currents 



which bring food and oxygenated water into the clam and carry wastes away, 
any impairment of ciliary function by a pollutant would be detrimental to 
the clam. , 

Once the rapid assessment technique had been used to determine which 
water quality factors had the greatest effect on fingernail clam gills, two 
of the water quality factors were selected for further testing using acute 
and chronic bioassay methods. It was possible that one level of a water 
quality factor would elicit a response from a gill preparation, but not af- 
fect the intact organism, where additional homeostatic mechanisms were opera- 
ting. The acute and chronic bioassay methods were developed as part of this 
research, and we also investigated several alternative methods for determining 
when the clams had died. 

Collection of Fingernail Clams 

Fingernail clams were collected from the Keokuk Pool of the Mississippi 
River using an 18-foot boat equipped with a crane and a Ponar grab sampler 
(Figure 3) . Fingernail clams were separated from the mud by pressure-sieving 
the Ponar samples through a 30-mesh screen with a 12-volt battery-operated 
water pump. The clams were carried to the laboratory in 37-liter plastic 
coolers equipped with aerators and half-filled with Mississippi River water. 
Approximately 100 gallons of river water was pumped into a tank and brought 
back to the laboratory at Havana, Illinois. 

Rapid Screening Methods 

Clams used in the rapid testing apparatus at Southern Illinois University 
were transported from Havana by truck or shipped via parcel post in plastic 



10 




Figure 3. Live fingernail clams, Musculium transversum , were obtained from 
the Keokuk Pool, Mississippi River, using a boat specially 
equipped with a crane and a Ponar grab sampler. Clams were 
separated from the mud by pressure sieving the Ponar samples 
through a 30-mesh screen mounted on the side of the boat. 



11 



bags or jars surrounded by styrofoam insulation to minimize temperature 
changes. The clams were delivered within 3 to 5 days of capture. 

Clams used in the cilia monitoring apparatus were divided into small 
(1 to 5 mm shell length) and large (6 to 11 mm shell length) size classes 
and kept in separate tanks. They were acclimated at least one week in 
invertebrate physiological solution in two Instant Ocean aquaria (tempera- 
ture 17 C and pH 7.8 to 8.2). Two of the chemicals tested, sodium and 
potassium, are components of the standard physiological solution used to 
maintain the clams and to bathe the isolated gills. In experiments where 
potassium or sodium was the test material, physiological solution was pre- 
pared without the test material. Reagent grade salts were then added to the 
solution to produce the desired concentration of the test material. 

The effects of suspended particles on fingernail clam gills were 
determined by measuring the rate of transport of particles across isolated 
clam gills maintained in petri dishes. The clam gills were observed under 
a microscope, and the movement of particles across a known distance in the 
microscope field was timed. The effects of low dissolved oxygen concentrations 
in combination with suspensions of illite clay and silica flour were also 
determined. 

The effects of the following 13 factors and factor combinations on the 

ciliary beating rate of fingernail clam gills were determined: 

Temperature Potassium Chloride Ammonium Chloride 

Dissolved Oxygen Potassium Cyanide Raw Illinois River Water 

and Sediment 
Sodium Cyanide Lead Nitrate 

Low Dissolved Oxygen and 
Sodium Nitrate Copper Sulfate Ammonium Chloride 

Sodium Sulfate Zinc Sulfate 



12 



The apparatus for monitoring ciliary activity of clam gills was 
specially constructed for this research (Figure 4) . The apparatus 
consists of two coupled microscopes with an aluminum scanning stage 
which holds two petri dishes , each containing a gill preparation. Water 
from a constant-temperature bath circulates through coils embedded in the 
walls of the stage in order to control the temperature in the petri dishes. 



1 rl ii ? 




Figure 4. Apparatus for measuring the effects of water quality factors on 
the ciliary beating rate of clam gills. 

A: Analog to digital converter on the stroboscopic light controller. 
B: Stroboscopic light reflector leading to two light rods which 

transmit light to the stages of the microscope. 
C: Motorized scanning stage with a hollow core for the circulation 

of water from a constant temperature bath. 
D: Push-button control for the motorized stage. When a button is 

depressed, the stage will automatically return to a preselected 

point. 
E: Digital display of ciliary beating rate. 



13 



Temperatures and dissolved oxygen concentrations in the petri dishes are 
monitored by thermistor meters and membrane electrodes. A continuous flow 
of standard physiological solution or solution to which test chemicals have 
been added can be maintained across the petri dishes by means of metering pumps. 

Twelve sets of measurements are made at each observation time on each 
gill. The platform positions are initially adjusted so that comparable areas 
on two gills are examined at the same time, one gill appearing in the left 
microscope and one in the right. The scanning stage is controlled by a 
servo-mechanism capable of moving in an X and Y direction. Each point has 
its own X and Y coordinates and is independently isolated from the coordi- 
nates of the other eleven points. Once the twelve positions are locked into 
the stage-controlling mechanism at the beginning of the experiment , the ob- 
server can return to any of the twelve positions simply by pushing a button. 
The stage can be automatically returned to within ten ym of the original point, 

A calibrated stroboscopic light serves as a substage light source for 
both microscopes. The light is divided and transmitted to the microscopes 
by means of a silver-coated Y-shaped Pyrex glass rod. 

The rate of ciliary beating of lateral cilia (in beats per second) is 
measured by manually synchronizing the rate of flashing of the light with 
the rate of beating of the lateral cilia, which beat in a metachronal pattern. 
Synchronization is achieved when the metachronal wave appears to stand still. 
The beating rate is shown on a digital display. 

Gills from a clam were isolated under a 30-power dissecting microscope 
and pinned to a rubber mat in a petri dish. In each experiment, 14 to 16 
gill preparations were tested. Figure 5 is a diagram of generalized clam 
anatomy (the detailed anatomy of the fingernail clam differs somewhat from 



14 



A Ad 




Figure 5. Generalized clam anatomy, showing the visceral ganglion (VG) and 
the gill (Gi) , which are used in the cilia monitoring apparatus. 
(The detailed anatomy of the fingernail clam differs from that in 
the diagram.) The enlarged diagram of section A-B across a single 
gill filament (GF) shows the lateral cilia (LC) . It is the beating 
rate of the lateral cilia which is monitored in the apparatus. The 
anterior adductor muscle (AAdM) and posterior adductor muscle 
(PAdM) close the shells. The paired palps (P) are oxygen sensors. 
The central nervous system consists of the pedal ganglion (PG) , 
cerebral ganglion (CG) , and visceral ganglion (VG) . These ganglia 
are interconnected by the cerebral-pedal connective (CPC) and the 
cerebral-visceral connective (CVC) . In cross section each gill 
appears in the form of a narrow "W". The inner and outer surfaces 
are made up of gill filaments (GF) . Within the afferent branchial 
blood vessel (ABV) , the branchial nerve (BN) , which arises from 
the visceral ganglion, distributes its axons within the gill 
filament. The lateral cilia (LC) produce water currents, while 
the latero-frontal cilia (LFC) remove particles from the water. 



15 



that in the diagram) showing the visceral ganglion and the gill. Section 
A-B across a single gill filament shows the location of lateral cilia (LC) , 
latero-frontal cilia (LFC) , and frontal cilia (FC) . The action of the cilia 
is partially controlled by the branchial nerve (BN) and visceral ganglion 
(VG) . Figure 6 is a scanning electron micrograph of gill filaments from 
the blue mussel, Mytilus edulis . showing the gill filaments (GF) , lateral cilia 
(LC) , latero-frontal cilia (LFC) , and frontal cilia (FC) , which are similar 
to those in the fingernail clam. Responses of the lateral cilia to water 
quality factors were measured in this study. 




Figure 6. Scanning electron micrograph of gill filaments from the blue mussel, 
Mytilus edulis , showing the gill filaments (GF) , lateral cilia (LC) , 
latero-frontal cilia (LFC) , and frontal cilia (FC) , which are 
similar to those in the fingernail clam. 



16 



Acute Bioassay Methods 

Clams used in the acute static bioassays were maintained in river water 
until they were acclimated to the test water. The clams were acclimated to 
the test waters as suggested by the Committee on Methods for Toxicity Tests 
with Aquatic Organisms (1975). 

Both adult clams (clams longer than 5 mm) and juvenile clams (5 mm and 
shorter) were used in bioassays. The bioassays will be identified consistently 
throughout the text by a J for tests conducted with juveniles and A for tests 
conducted with adults. Al is the first test conducted with adults, A2 the 
second test, etc. 

Most of the clams were exposed to a controlled light-dark cycle during 
acclimation and testing. During tests A2, A4, J2, and J3 the cycle was 14 
hours of light and 10 hours of dark (Table 1). For the low-temperature test 
J4, the cycle was 10 hours of light and 14 hours of dark (Table 1) which 
approximates a natural winter cycle. The light sources were 40-watt cool- 
white fluorescent bulbs. Clams in acute tests Al and Jl were not exposed to 
a controlled light-dark cycle, but to diffuse room light. 

The dilution waters used in these experiments were from two different 
sources: unchlorinated well water from the Department of Conservation in 
Havana, Illinois, and reconstituted water (Marking, 1969). The chemical 
characteristics of the dilution waters were determined from samples collected 
at the beginning of each bioassay (Table 1). A logarithmic series of test 
solutions was prepared by diluting aliquots of known stock solution to 3 liters. 

Heavy metal concentrations in the dilution water and test solutions were 
measured by either atomic absorption or flame photometry. Hardness, ammonia. 



17 



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18 



nitrite, nitrate, and phosphate were measured by standard methods (American 
Public Health Association, 1976). To measure potassium, water samples were 
collected 3 times during each bioassay and analyzed by flame photometry, 
except in acute tests Al and Jl, where composite samples were used. In the 
ammonia tests, samples were collected for ammonia analysis from each test 
chamber 3 times weekly. Total ammonia concentrations were measured using a 
specific ion electrode and converted to undissociated ammonia using test pH 
and temperature and tables presented by Thurston et al. (1974). 

Test chambers for the acute static bioassays consisted of 3.78-liter 
glass jars, which were immersed in a temperature-controlled water bath. Ten 
or 20 clams were contained in a petri dish (100 mm in diameter) covered with 
a plastic snap-on lid. A 50-mm diameter hole was cut in the center of the lid 
allowing circulation of water to the clams while preventing the clams from 
crawling out. One petri dish was placed in each test chamber. The petri dishes 
could be removed and examined with a dissecting microscope without greatly 
disturbing the clams. Clams were not fed during the test. Only clams that 
were actively siphoning or moving were selected for the tests. 

Criterion for death was determined during the first adult and juvenile 
static bioassays, tests Al and Jl, respectively. Clams which gaped, or which 
did not withdraw their feet when prodded, were removed from the test concen- 
trations and placed in clean water. They were checked 24 hours later for 
signs of recovery. Clams that recovered were not used in determining the 
concentrations lethal to 50 percent of the exposed clams (LC50) . 

Observation times varied slightly from one test to another, but in general 
the clams were checked for mortalities once every 24 hours for the first 96 
hours, once every 48 hours from hours 96 to 240, once every 72 hours between 



19 



hours 240 and 456, and every 96 hours for the remainder of the test. Tem- 
perature and dissolved oxygen were measured at each mortality check and pH 
and total alkalinity were determined every other mortality check by 
standard methods (American Public Health Association, 1976). 

For the acute bioassays the LCSO's and their confidence limits were 
determined and statistically analyzed by the method of Litchfield and Wilcoxon 
(1949). To adjust for mortality in the control, Abbott's formula, given 
below, was used as suggested by the American Public Health Association (1976). 

p'-c 
Abbott's Formula: P = -^ — , where: 

P = corrected mortality 

p' = observed mortality 

c = control mortality 

For example, with a control mortality of 20 percent and an observed test 
mortality of 60 percent, the corrected mortality would be 50 percent. Ob- 
served mortalities were adjusted in this fashion prior to applying the Litch- 
field and Wilcoxon (1949) method. A test mortality less than the control 
mortality was considered to be zero. 

Acute toxicity curves were plotted according to Sprague (1973). LC50's 
were plotted with time to 50 percent mortality on the vertical axis and con- 
centration on the horizontal axis. The resulting curve is the acute toxicity 
curve. The point on the concentration axis where the curve becomes asymptotic 
to the time axis defines the lethal threshold or the point where no more or- 
ganisms are dying and the remaining organisms could presumably live indefinitely. 
A threshold was not considered valid unless the asymptotic portion of the curve 
was maintained for a period of time equivalent to at least 20 percent of the 



20 



time that was required to reach the asymptote, as suggested by Ruesink and 
Smith (1975). If the asymptote was approached but not completely attained, 
or if it was reached but did not meet the time criterion indicated above, it 
was considered a probable lethal threshold. 

Toxicity curves were compared to determine relative toxicity of different 
factors and to determine how fast the factors induced effects. 

Chronic Bioassay Methods 

Clams used in the chronic bioassays began acclimation to unchlorinated 
well water immediately upon arrival at a laboratory provided by the Illinois 
Department of Conservation at Havana. Acclimation procedures followed recom- 
mendations by the Committee on Methods for Toxicity Tests with Aquatic Organisms 
(1975). The clams were exposed to natural daylight coming through north and 
west windows on the sides of the building. 

The test chambers were 37.8-liter glass aquaria with outlets arranged so 
that the aquaria contained 23 liters of water (Figure 7) . A modified proportional 
diluter (Mount and Brungs, 1967) was used to deliver a logarithmic series of 
test solutions to five aquaria and unchlorinated well water alone to the 
control aquaria (Figure 7). The effects of ammonia and potassium on fingernail 
clams were tested with this apparatus. 

The effects of raw Illinois River water and sediment were tested by 
maintaining fingernail clams in cages in the Illinois River, and by exposing 
fingernail clams to both raw river water and river water diluted with well 
water in a laboratory located next to the Illinois River at Havana and provided 
by the Illinois Department of Conservation, Fisheries Division. River water 
containing suspended sediment was pumped into a reservoir in the laboratory, 



21 




Figure 7. Toxicant diluters which 
delivered test solutions 
or well water to aquaria 
which served as test 
chambers for the chronic 
bioassays. 



where a circulating pump kept the sediment in suspension and provided 
sediment-laden water to the diluter. 

During preliminary testing of ammonia, we found that increasing con- 
centrations of NH.Cl reduced the pH of the test solutions, thus necessitating 
the addition of NaOH in proportion to the concentration of NH.Cl. This was 
accomplished by adding a second metering system on the diluter which once 
each cycle delivered a measured volume of a concentrated NaOH solution to the 
toxicant -mixing chamber. The NaOH metering system was calibrated to deliver 
4 ml of a NaOH solution which varied in concentration between 0.025 M and 



22 



0.125 M depending on the pH of the test chambers. In addition we found that 
cleaning the entire testing system once a week aided in pH control. 

A feeding system was also incorporated into the proportional diluter 
so as to deliver a measured amount of food once per diluter cycle. The food 
suspension was prepared as suggested by Biesinger and Christensen (1972). 
Preliminary testing showed the optimum feeding rate to be 0.80 ml of the 
food suspension per liter of test water. 

In the chronic test 20 clams were used per petri dish with two petri 
dishes in each chamber. Survival and growth of the clams was checked once 
every two weeks. Growth was determined by measuring the maximum length of 
the shell to the nearest 0.1 mm with an ocular micrometer. Temperature and 
dissolved oxygen were measured 5 times a week, pH three times a week, and total 
alkalinity once a week except in two tests where pH was checked five times a 
week. Free carbon dioxide concentrations were calculated from the pH and 
alkalinity data using the indirect method of Rainwater and Thatcher (1960). 

Data from the chronic bioassays were used to determine maximum acceptable 
toxicant concentrations (MATC) as suggested by the American Public Health 
Association (1976). MATC's were computed using both mortality and growth as 
responses. Replicate mortalities in each test chamber were subjected to 
analysis of variance, ANOVA (Steel and Torrie, 1960). When treatment effects 
were indicated by ANOVA, the means of these effects were subjected to the 
Newman-Keul ' s multiple-range test (Steel and Torrie, 1960). Pooled clam lengths 
from each test chamber were also subjected to analysis of variance and the 
Newman-Keul ' s multiple-range test. Data collected from test chambers showing 
mortalities significantly higher than the controls were not used in the 



23 



analysis of clam lengths. All differences were considered statistically 
significant at a probability of p = 0.05. 

Elemental Analysis of Shells 

Dr. Judith Murphy, Director of the Center for Electron Microscopy at 
Southern Illinois University — Carbondale, determined the elemental composi- 
tion of shells from fingernail clams which had been chronically exposed to 
raw Illinois River water or to ammonia. She used X-ray microprobe techniques 
to determine relative amounts of specific elements in specific regions of 
the shells, starting from the hinge area and proceeding outwards to the 
shell margin. Since the clam grows by adding to the shell at the margin, 
chronological changes in the composition of the shell were determined. 



24 



RESULTS AND DISCUSSION 

Water Quality in the Illinois River in the 1950' s 

When the fingernail clams died out in portions of the Illinois River in 
the mid-1950' s, relatively few analyses of toxic substances in Illinois River 
water were being made and the analytical methods were not sensitive enough 
to detect concentrations of parts per million or parts per billion which can 
affect aquatic organisms. Some water quality factors which are generally 
considered non-toxic were measured by the Illinois State Water Survey at 
Peoria during the 1950' s. These data show that sodium and chloride concen- 
trations in the Illinois River have increased since 1950, partly due to the 
increasing use of salt to melt ice on streets and highways. 

Dr. Ronald Flemal, Associate Professor of Geology, Northern Illinois 
University, speculated in a letter dated 5 May, 1976 that the potassium 
concentrations in the Illinois River may have increased during the same 
period also: 

The Water Survey . . . l\\asj two stations on the main stem (Peoria 
and Meredosia) , and potassium analyses only at Peoria for the period 
1966-71. . . . 

In the absence of actual potassium data, I am not sure if we can say 
much positive about past trends in potassium concentrations; the best 
we can do is speculate. . . . 

The chloride data display an obvious upward trend, with a particularly 
significant upward step in 1962-63. Chloride enters the environment 
in many ways .... One of the more important of these is as potassium 
chloride fertilizer, and the 62-63 period is about when potassium chloride 
fertilizer became widely used. One might expect therefore that potassium 
concentrations (at least during the fertilizing season) turned upward 
in mirror to the chloride concentrations. 

A somewhat similar case can be made from the "sodium" data. I give 
the data in quotation marks because previous to 1966 the Water Survey 
determined sodium by calculation. This means that the reported values 
of sodium are actually sodium and potassium plus assorted lesser cations. 
Two observations then make the case: (1) the sodium data closely 



25 



correspond to the chloride data, and (2) the differential between 
the chloride and sodium lines decreases in 1966 when the potassium 
is actually split out from the "sodium." These two imply that the 
potassium is a significant part of the "sodium" previous to 1966 and 
that as the "sodium" curve rises, so also ought a potassium curve 
have risen if it could have been calculated. 

These are admittedly not the strongest of arguments for an 
historical change in potassium concentrations, but they are the 
best I can make at the moment and without further investigation. 

Potassium is nontoxic to fish, but Imlay (1973: 97) demonstrated that 
potassium concentrations of 11 mg/1 killed several species of mussels 
in 1-2 months. 

Mills et al. (1966: 9) provide a table, compiled from various sources, 
of minimum dissolved oxygen levels near the surface in the channel of the 
Illinois River from 1911 through 1965. In 1950, the average dissolved 
oxygen concentration was 4.0 (range 2.9-5.3). There is a gap in the data 
until 1964, when the average oxygen concentration in the same reach was 2.7 
mg/1 (range 2.0-5.3). Conditions were even worse in 1965, when the average 
was 2.3 mg/1 and the range 1.0-5.6. The minimum dissolved oxygen levels 
in the Illinois River evidently declined after 1950. 

To summarize: (1) There are few historical data available on concen- 
trations of toxic substances in the Illinois River in the 1950' s, (2) available 
data show that concentrations of sodium, chloride, and possibly potassium 
increased in the river in the 1950' s, and (3) dissolved oxygen levels in the 
river may have decreased in the 1950* s. 

Comparison of Water Quality in the Mississippi and Illinois Rivers in 1975 

Since fingernail clams have never recolonized the Illinois River since 
the die-off in the 1950' s, comparison of present water quality in the Illinois 
River with water quality in the Mississippi, where the clams are still abun- 
dant, might indicate which water quality factors are affecting the clam. The 



26 



Illinois Environmental Protection Agency (lEPA) currently analyzes water 
samples from the major rivers of the state for twenty-eight water quality 
factors. 

Tables 2, 3, and 4 show mean, median, and maximum values of sixteen water 
quality factors which occurred at higher levels in 1975 at four sampling 
stations on the Illinois River than at a sampling station on the Keokuk 
Pool, Mississippi River. The four stations are in a reach of the Illinois 
River where fingernail clams died out in the 1950' s. One other factor, 
dissolved oxygen, was generally lower in the Illinois River than in the 
Mississippi River. 

The following four factors regularly occurred at substantially higher 
concentrations in the Illinois River than in the Mississippi River and are 
known to be toxic to fish: ammonia, lead, fluoride, and methylene blue 
active substances. Nitrite is less toxic to fish than ammonia, but more 
toxic than nitrate. Unfortunately, nitrite and nitrate are reported as a 
combined value by the Illinois Environmental Protection Agency. Counts of 
fecal coliform bacteria were much higher in the Illinois than in the Missis- 
sippi, but clams feed on bacteria, so it is not likely that increased bac- 
terial populations adversely affected fingernail clams. The maximum water 
temperature in midsummer of 1975 in the Keokuk Pool, Mississippi River, dif- 
fered from the maximum in the Illinois River by only 1° F (Table 4) . Although 
the median and mean water temperatures were higher in the Illinois than in 
the Mississippi (Tables 2 and 3) , the range of the fingernail clam Musculium 
transversum extends into southern parts of the United States, where the mean 
water temperature equals that in the Illinois River. Total dissolved solids, 
chloride, phosphorus, and sulfate were higher in the Illinois River than in 
the Mississippi, but these factors are not considered toxic to fish. 



27 



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CO O 



,0 CO 

o <u c 

B -H 



4J ^ 

CO M (U 

CO 2" 
(U o 



U > 



g S 



CO X 

CO X) 



30 



It is difficult, if not impossible, to relate the die-off of fingernail 
clams in the Illinois River to water quality factors, because the water 
quality requirements of fingernail clams are not known. The next two sub- 
sections report the results of our efforts: (1) to develop rapid methods 
for assessing the effects of water quality on fingernail clams and (2) to 
develop standard acute and chronic bioassay procedures, including a reliable 
indicator of death, using clams as test organisms. 

Reliability of the Rapid Screening Methods 

At 21° C with an oxygen concentration of 6.5 mg/1 and a pH of 7.6, the 
beating of the lateral cilia on the excised gill preparation was rapid, well 
coordinated, and fairly constant after two hours of equilibration. The gill 
preparation seemed to be fairly robust, because normal ciliary activity was 
maintained for at least eight days. The standard deviations of the mean 
ciliary beating rates are reported in the tables or plotted on the graphs which 
follow in the rest of the results section. Only the standard deviations in 
the positive direction are plotted (as one half of a bracket) , to avoid 
cluttering the figures. In general the standard deviations were quite small 
and uniform. Even small changes in the levels of various water quality fac- 
tors produced marked changes in the average ciliary beating rates, with 
little or no overlap of the standard deviations. 

The particle transport rate across the gills proved to be similarly re- 
liable and sensitive. 

The sensitivity of the rapid method is compared to the sensitivity of 
the chronic bioassay method in the results subsections on potassium and 
ammonia. 



31 



The Gaping Response as an Indicator of Death 

Death was best indicated by the gaping response. Gape was due to 
relaxation of posterior and anterior adductor msucles , after which the 
elastic external ligament forced the shells open. The clam did not respond 
when the body was prodded through the open shell and was incapable of keeping 
the shell closed when the valves were forced together. Clams failed to re- 
cover after exhibiting this response. 

The death criterion was verified in tests A2 and J2. Clams exhibiting 
the gaping response were removed and placed in clean water. In both of these 
tests, 100 percent of the clams failed to recover after exhibiting this 
response. 

Some adults exhibited a partial gape which was not a valid death indi- 
cator. A partial gape was characterized by a valve separation of 1 mm or 
less in conjunction with a lack of response to prodding. Clams recovered 
from this condition if placed in clean water. Juvenile clams never exhibited 
a partial gape. 

Other responses that the clams exhibited that were not death indicators 
were immobilization, cessation of heart beat (visible through the transparent 
shell), contraction of body toward the umbo region, and lack of response to 
prodding of the extended foot or siphon upon which the valves were tightly 
closed. 

Acute and Chronic Bioassay Methods with Juvenile and Adult Fingernail Clams 

Two types of bioassay methods were used in this research: the acute 
static bioassay in which the clams were not fed, and the chronic bioassay in 
which the clams were fed and effects of the toxicant on growth were monitored. 



32 



The chronic bioassay was the preferred method of testing M. transversum for 
several reasons. The slow response of the clam is a primary consideration. 
Sprague (1973) stated that acute bioassays should not be terminated until the 
toxicity curve becomes asymptotic to the vertical axis and thus indicates a 
lethal threshold. Only adult test A2 (Figure 22 ) showed a valid lethal 
threshold after 240 hours (10 days) of exposure. Acute static tests J3 and J4 
(Tables 16 & 18) were extended beyond 600 hours (25 days) without demonstrating 
lethal thresholds. In general, the main advantage of using acute bioassay 
techniques is reduced testing time, e.g. in most fish bioassays a lethal 
threshold can be reached in 24 to 48 hours. This advantage was not evident 
in the acute tests with Musculium transversum . On the other hand, the species 
seems to be ideally suited for chronic bioassay. A properly conducted chronic 
bioassay should expose the organism to the toxicant for an entire life cycle. 
M. transversum , under optimum conditions, can complete an entire life cycle 
in 33 days (Gale, 1969). 

There were certain deficiencies in the chronic testing system. These 
are related mainly to improvements that are needed in the culture method. 
Gale (1969) stated that 5 mm was the minimum length of clam in which embryos 
normally occur. The best growth that was achieved in the chronic bioassays 
was in potassium test K2 where clams in one test chamber grew an average of 
1.4 mm in 28 days (Figure 26 ). This growth rate needs to be nearly doubled 
if reproduction is to be obtained within the optimum 33 days. The reasons 
for the reduced growth rate are unknown and experiments are currently being 
conducted to make improvements in the culture system. 

Toxicity curves of adults and juvenile clams under similar test condi- 
tions were compared (Figures 21 and 22 ) and were significantly different 
(p=0.05). Adults responded 5 to 1.6 times faster than the juveniles to 



33 



potassium. A lethal threshold was obtained at least 150 hours faster in the 
adult test than in the juvenile test (Figure 22 ). 

The most important reason for using M. transversum as a bioassay organism 
relates to its paramount ecological importance, as described in the Introduc- 
tion and Background. 

The responses of clams to light, raw Illinois River water, cyanide, low 
dissolved oxygen concentrations, and six of the sixteen factors which oc- 
curred at higher levels in the Illinois River than in the Mississippi River 
are described in the subsections which follow. 

Response of Clams to Light and Darkness 

Volumes have been written on the role of light and darkness in con- 
trolling physiological processes in both plants and animals (for example, Beck, 
1963) . It was important to determine the effects of light and darkness on 
fingernail clams. The lighting at the laboratory could then be controlled 
to avoid confounding the effects of lighting with the effects of the water 
quality factors. 

Figure 8 shows that the ciliary beating rates of gills from two Sphaeri- 
acean clams ( Musculium transversum and Corbicula manilensis ) and the unrelated 
intertidal mussel, Mytilus edulis, were inhibited by light from a fluorescent 
desk lamp containing two Sylvania F-15/T8 CW tubes positioned about 0.5 m from 
the gill preparations. Noticeable inhibition occurred after 1 to 2h hours of 
exposure to light and maximum inhibition occurred after 2 to 6 hours' exposure. 
When the gill preparations were returned to darkness, the ciliary beating 
rates returned to normal after 2 to 4 hours, with the exception of large Mus- 
culium transversum , whose ciliary beating rates had not recovered to normal 
by the time the experiment was ended after 4 hours exposure to darkness. The 



34 




Figure 8. Light inhibits the beating of lateral cilia on the gills of two 

Sphaeriacean clams ( Musculium transversum and Corbicula manilensis) 
and the unrelated intertidal mussel, Mytilus edulis. Beating rates 
recovered when the light was turned off. 



response of small fingernail clams to light was more similar to that of the 
intertidal mussel, than to the large fingernail clams. 

Musculium transversum lives in almost perpetual darkness in or on the 
bottom of the turbid Mississippi River. However, there are a few occasions 
when light does reach the bottom, such as in mid-winter, under ice, a time 
when the clam is normally dormant in Keokuk Pool, Mississippi River. Perhaps 
the inhibition of the cilia by light is a protective response, should the clam 
be exposed to falling water levels or to the air. The shell of the fingernail 
clam is certainly translucent and practically transparent. It would be inter- 
esting to determine whether the clam feeds more actively at night when it 



35 



lives in very shallow, or clear water. Perhaps the increasing exposure to 
daylight, which would result from decreasing water levels over shallow 
areas, would stimulate the clams to burrow into the mud and cease normal 
activity (including ciliary activity) thus protecting them from desiccation. 

All subsequent experiments with water quality factors were conducted 
in a photographic darkroom, because the maximum ciliary beating rate was ob- 
tained in darkness. The only illumination was provided by a photographic 
safe light with a Kodak lA filter. The safe light did not influence the 
ciliary beating rate. Stroboscopic measurement of the ciliary beating rate 
took only a few seconds, and was not long enough to cause inhibition of the 
cilia. Intact clams were exposed to natural light from windows or timer- 
controlled lights during the chronic and acute bioassays, except for acute 
tests Al and Jl where they were exposed to diffuse, low-intensity room light. 

Response of Clams to Temperature 

Gill preparations from the intertidal mussel, Mytilus edulis , maintained 
their ciliary beating rates over a broader temperature range than gill prepara- 
tions from fingernail clams or Asiatic clams (Figure 9) . It is not surprising 
that the intertidal mussel shows such a broad temperature range, for it must 
withstand daily exposure to the hot sun when the tide is out, followed by 
sudden immersion in cold sea water when the water returns. Small fingernail 
clams have a broader temperature range than large ones, and the Asiatic clam 
appears to have the narrowest temperature tolerance, at least as measured by 
the ciliary beating response. 

We have found that fingernail clams begin to grow rapidly when water 
temperatures in the Keokuk Pool, Mississippi River rise above 11-13 C. Figure 9 
shows that the ciliary beating rate of the fingernail clam substantially . 



36 



12 1 



• - MYTILUS ECilLiS 

n = nuscuLiuM tr an sy ersum (small) 

0=: WUSCULIUM TR AN SY ERSUM (lARGE) 

A= cor bicula man I lens is 




TEMPERATURE (C°) 



Figure 9. Ciliary beating response of gills from large and small fingernail 

clams ( Musculium transversum ) , the Asiatic clam ( Corbicula manilensis ) 
and the blue mussel (Mytilus edulis ) to temperature. 



increased above 14-15° C, so the field results appear to corroborate the labora- 
tory findings. The laboratory results also suggest that the ciliary function 
of fingernail clams would be drastically reduced if water temperatures in- 
creased above 30° C. 

It is surprising that the gills from Asiatic clams showed a maximum 
beating response at 20°C, and a beating rate of practically zero at 30°C, 
although Asiatic clams are known to inhabit thermal effluents which exceed 
30°C. (Personal communication, February 10, 1978, Mr. Herbert Dreier, Aquatic 
Biologist, Illinois Natural History Survey.) Mattice and Dye (1976) re- 
port that the upper temperature tolerance is 34° C, for Asiatic clams 



37 



acclimated to 30°C. However, all gill preparations used in our experiments 
came from clams which had been acclimated to water temperatures of 17°C, and 
it is likely that the optimum temperature for the ciliary beating response 
would be shifted toward higher temperatures, if the clams were acclimated to 
higher temperatures. Additional experiments would have to be performed to 
determine the acclimation range of these three species of clam, and the 
relationship between the ciliary beating response and acclimation temperature. 
Figures 10 and 11 show that the ciliary beating rate of gills from both 
large and small fingernail clams is proportional to the water temperature. 
The small fingernail clams showed a greater response to increased water 
temperature than to large clams. When the temperature was increased from 18 
to 21°C, the ciliary beating rate of small clams increased from 9 to 20 
beats per second, whereas the ciliary beating rate of gills from large clams 
increased from 8 to 11 beats per second for just a few seconds. The effects 
of potassium cyanide and sodium cyanide are discussed in another subsection. 

Response of Clams to Dissolved Oxygen 

Figure 12 shows that the higher the concentration of dissolved oxygen 
in the water perfusing the gill preparation, the greater the ciliary beating 
rate. The ciliary beating rate of gill preparations from large clams rapidly 
declined to zero when the dissolved oxygen concentration was reduced to 2 
parts per million. Gill preparations from small clams showed a similar 
response, although the results are not plotted in Figure 12 for the sake of 
clarity. It is unlikely that dissolved oxygen concentrations of 2 parts per 
million or lower would immediately kill fingernail clams in nature. Most, 
or perhaps all clams can switch from aerobic to anaerobic metabolism when their 
shells are closed. We did not conduct any acute or chronic bioassays to 



38 



MusculiumC large ) 
•039ivig/i K 



3o- 



^fWpt«^WW^W2 5°C 



20 



(0 

q: 

UJ 
Q. 

(0 
h 
< 10 

LU 
DQ 




Figure 10. Beating 
rate of cilia on gills 
from large fingernail 
clams is proportional 
to the increase in water 
temperature. All large 
clams were maintained in 
a potassium concentration 
of .039 mg/1 during the 
experiments in order to 
maintain normal ciliary 
activity. Addition of 
98 mg/1 sodium cyanide 
at time A inhibited 
the cilia, even at a 
stimulatory temperature. 



Musculium(smal I) 
39mg/| 




^26 C 


Figure 11. Beating 


4l 0^ 


rate of cilia on gills 


j4*25"C 


from small clams is 




proportional to the 


U24°C 


increase in water tem- 


perature. All small 




clams were maintained 




in a potassium concentra- 




tion of 39 mg/1 during 


XX23°C 


the experiments in 


r^ 


order to maintain normal 


il2 2°C 


ciliary activity. 


r-A-^*- *- 


Addition of 98 mg/1 


r^21°C 


sodium cyanide or 130 mg/1 


potassium cyanide at time 




A gradually inhibited 


r o^ 


the cilia, even at a 


Ar26X + 


stimulatory temperature. 



g/| NaCN 



36 54 

SECONDS 



72 



39 




Figure 12. The ciliary beating rate of gills from large and small fingernail 

clams increased as the oxygen concentration of the water increased. 
The ciliary beating rate of both large and small clams rapidly 
declined to in water containing 2 mg/1 or less oxygen. (The 
response of small clams to decreased oxygen is not plotted for 
the sake of clarity.) 



determine how long intact fingernail clams could resist dissolved oxygen 
concentrations of 2 parts per million or lower. Fingernail clams are found 
in the Des Plaines River, where the oxygen concentrations probably reach low 
levels due to oxygen-demanding wastes (personal communication, October 15, 1976, 
Mr. Thomas Butts, Chemist, Water Quality Section, Illinois State Water Survey). 

A common feature of the anatomy of many species of clams is a pair of 
palps located near the mouth (Figure 5). The palps are oxygen-sensing organs, 
which apparently stimulate the lateral cilia to beat faster when the oxygen 
content of the water is increased. When the palps are removed, the gill 




2 I 10 
CONCENTRATION OF DISSOLVED OXYGEN (mg/l) 



Figure 13. The ciliary beating rate of gills from large and small fingernail 

clams ( Musculium transversum ) , Asiatic clams ( Corbicula m.anilensis ) , 
and the blue mussel ( Mytilus edulis ) declined when the oxygen 
level was reduced from 10 to 2 mg/l, and recovered when the oxygen 
level was restored to 10 mg/l. When the oxygen-sensing palps were 
removed, the gills responded as though they were in oxygen- 
deficient water, although the ambient oxygen concentration was 
maintained at 10 mg/l. 



preparations behave as though they were in water containing little dissolved 
oxygen, even though oxygen levels in the water are maintained at 10 parts 
per million (Figure 13). 

Response of Clams to Sodium Nitrate and Sodium Sulfate 

Concentrations of nitrite-nitrate, sulfate, and total dissolved solids 
in the Illinois River were greater than in the Mississippi River (see Tables 2-4) 
The purpose of exposing gill preparations to sodium nitrate and sodium sulfate 



41 



was to determine whether any of the above three factors could have affected 
fingernail clams. Moreover, the metals lead, copper, and zinc were added in 
the form of nitrates or sulfates, so it was important to determine the relative 
contribution, if any, of the sulfate and nitrate portion of the salts to the 
toxicity of the solutions. 

Table 5 shows that the highest concentrations of sodium nitrate and sodium 
sulfate tested had no effect on the ciliary beating rates of gills from large 

or small clams. The concentrations of the metals are given as gram atomic 

_3 
weights, and are converted to mg/1 as follows: sodium (10 x the gram atomic 

_3 
weight of sodium, or 22.9 mg/1), nitrate (10 x the gram ionic weight of 

_3 
nitrate, or 62.0 mg/1), and sulfate (.5 x 10 x the gram ionic weight of 

sulfate, or 48.0 mg/1). The Illinois Environmental Protection Agency expresses 

nitrate-nitrite concentrations as nitrogen, in mg/1. The maximum nitrate 

concentration we tested, 62.0 mg/1, is equivalent to 18 mg/1 as nitrogen. 

The maximum nitrate-nitrite concentration (as nitrogen) shown in Table 5 for 

the water quality sampling stations in the Illinois River is 7.6, so nitrate 

concentrations in the river certainly were not high enough in 1975 to impair 

the ciliary activity of fingernail clams. The maximum sulfate concentrations 

at the sampling stations in the Illinois River ranged from 101 to 155 (Table 5), 

considerably above the maximum sulfate concentration we tested, so sulfate 

cannot be ruled out as a factor which might affect fingernail clams in the river. 

The total dissolved solids (TDS) concentration in the Illinois River 

(Table 5) is determined by measuring the specific electrical conductivity 

of the water (in micromhos/cm) and multiplying by 0.6 (Illinois Environmental 

Protection Agency, 1976: ii) . The maximum conductivity reported for the 

Illinois River in 1975 can be computed by dividing the maximum total dissolved 



^2 

Table 5 . Effects of Sodium Nitrate, Sodium Sulfate, Lead Nitrate, 
Copper Sulfate, and Zinc Sulfate on the Average Ciliary Beating Rate of the 
Gills of Musculium transversum .^ 

Gram Atomic Average Rate of Beating (Beats/Sec.) 

Weight of Metal Sodium Lead Sodium Copper Zinc 
per Liter Nitrate Nitrate Sulfate Sulfate Sulfate 





Small 


Clams (< 5, 


. mm maximum 


shell len 


gth) 


(control) 


10.8+0.9 ^ 


11.5+0.6 


12.1+0.9 


13.1+0.5 


12.6+0.4 


10-12 


10.7+0.8 


11.3+0.4 


12.3+0.6 


10.5+0.4 


10.9+0.3 


10-11 


10.9+0.5 


10.5+0.3 


12.1+0.2 


7.8+0.8 


11.2+0.1 


10-10 


10.5+0.7 


9.8+0.2 


11.8+0.6 


7.0+0.1 


10.6+0.1 


10-9 


10.6+0.8 


6.8+0.2 


11.6+0.7 


6.1+0.5 


7.3+0.3 


10-S 


9.9+0.9 


6.1+0.3 


11.3+1.2 


4.1+0.2 


5.5+0.1 


10-7 


10.1+0.8 


4.2+0.2 


11.5+0.9 


2.1+0.1 


5.1+0.1 


10-6 


10.3+0.4 


3.5+0.4 


11.3+1.1 


1.8+0.3 


2.0+0.3 


10-5 


10.5+0.6 


2.7+0.6 


11.6+0.2 


1.2+0.6 


1.0+0.2 


10-^ 


10.9+0.5 


1.8+0.3 


11.5+0.3 


1.7+0.3 


0.8+0.1 


10-3 


10.6+0.8 


1.1+0.2 


11.9+0.9 


1.3+0.2 


0.6+0.1 



Large Clams (> 5.0 mm maximum shell length) 



(control) 
10-12 

10-11 
10-10 


9.8+0.5 

9.7+0.8 

9.6+0.7 

10.1+0.3 


9.6+0.8 
9.1+0.6 
8.1+0.4 
5.3+0.3 


10.1+0.9 
9.8+0.2 
9,9+0.4 

10.3+0.2 


9.6+0.3 
8.3+0.3 
6.1+0.2 
2.1+0.1 


8.9+1.5 
4.3+0.5 
3.8+0.8 
3.2+0.7 


10-9 


10.3+0.2 


2.1+0.1 


10.6+0.3 


1.2+0.1 


2.1+0.9 


io-« 


9.6+0.3 


1.0+0.2 


9.8+0.7 


0.8+0.1 


1.2+0.8 


10-7 


9.8+0.5 


0.8+0.1 


9.7+0.6 


0.3+0.1 


1.1+0.2 


10-6 


9.6+0.7 


0.6+0.2 


9.7+0.8 


0.2+0.2 


1.0+0.3 


10-5 


9.7+0.9 


0.7+0.3 


9.8+0.2 


0.3+0.3 


0.8+0.3 


10-^ 


9.8+0.3 


0.4+0.1 


10.3+0.3 


0.1+0.1 


0.2+0.2 


10-3 


10.1+0.6 


0.3+0.1 


10.6+0.4 


0.2+0.1 


0.3+0.1 



Each point is an average of 14 gills and 12 readings per gill, or a total 
of 168 observations. 

b 
Mean + standard deviation. 



43 



concentration of 440 mg/1 by 0.6, yielding 733 micromhos/cm. We did not 
measure the conductivity of our test solutions. It is possible to compute 
the approximate conductivity of solutions using methods given in Sawyer and 
McCarty (1967: 184-186) and tables in the Handbook of Chemistry and Physics 
(1962: 2692-2696). For example, the amount of sodium sulfate added to the 

strongest test solution would produce an approximate conductance of 111 

2 
micromhos/cm . This does not include the contribution made to total conduc- 
tance by other ions in the solution, but it indicates that the ionic strength 
of our test solutions was probably below the average and maximum ionic 
strength of the Illinois River. 

Response of Clams to Sodium Cyanide and Potassium Cyanide 

Figure 10 shows that the beating rate of cilia from the gills of large 
clams was inhibited by a cyanide concentration of 98 mg/1 (as CN, not as 
NaCN) , even at a temperature of 25 C, which would normally stimulate the cilia. 

Figure 11 shows that it took longer for a cyanide concentration of 98 
mg/1 to inhibit the ciliary beating rate of small clams than it did for large 
clams. A higher concentration (130 mg/1 as CN) inhibited the cilia of small 
clams sooner, but did not entirely prevent an initial increase in the beating 
rate due to the warm temperature (26 C) . 

Additional experiments would have to be conducted with cyanide to de- 
termine the threshold concentration for inhibition of cilia. 

Response of Clams to Lead Nitrate, Copper Sulfate, and Zinc Sulfate 

Since nitrate and sulfate did not have any effects on the ciliary beating 
rates of gill preparations, the marked effects obtained with solutions of 



44 



lead nitrate, copper sulfate, and zinc sulfate are attributable to the metals. 
Table 5 reports concentrations of the metals as gram atomic weights per liter 
(GAW/1) . The concentration or concentration range in which the ciliary beating 
rate was reduced to 50% of the normal level is given in milligrams per liter 
(mg/1) or micrograms per liter (yg/l) below: 

Large Clams Small Clams 

Lead .02 yg/1 2-20 yg/1 

Copper .0006 yg/1 .006-. 06 yg/1 

Zinc .00006 yg/1 .06-. 6 yg/1 

A concentration range is given in cases where the lower concentration produced 
less than a 50% reduction in the ciliary beating rate and the next higher 
concentration produced more than a 50% redrction. The concentration which 
would cause a 50% reduction would lie between the two concentrations actually 
tested. 

The concentrations which caused 90% reductions in ciliary beating 
rates are: 

Large Clams Small Clams 

Lead 2 yg/1 207 mg/1 

Copper .06-. 6 yg/1 63 mg/1 

Zinc .06-. 65 mg/1 .65 mg/1 

The gills from large clams are much more sensitive to the metals than gills 
from small clams; or stated another way, the clams become more sensitive to 
these metals as they grow older and larger. This is the reverse of the case 
with fish, where the juvenile stages are usually more sensitive to toxicants 
than the adults. 

In 1975, the average, median, and maximum concentrations of lead, copper, 
and zinc in the reach of the Illinois River between Creve Coeur and Havana and 



45 



in the Mississippi River at Fort Madison (Illinois Environmental Protection 
Agency, 1975, Volumes 2 and 4) were below the levels which would cause a 90% 
reduction in the ciliary beating rate of gills from small clams . Lead was 
below detectable limits in 3 samples taken from the Mississippi River at Fort 
Madison in 1975 (Illinois Environmental Protection Agency, 1975, Volume 4: 425) 
but averaged .01 to .02 mg/1 in 3 of the 4 sampling stations on the Illinois 
River (Illinois Environmental Protection Agency, 1975, Volume 2: 92, 93, 103, 
104). These concentrations would be sufficient to cause more than a 90% 
reduction in the ciliary beating rate of gills from large clams . The median 
concentration of copper in two of the Illinois River stations was slightly 
higher than in the Mississippi, and the maximum copper concentration of .02 
was the same in both rivers (Illinois Environmental Protection Agency, 1975, 
Volume 2: 92, 93, 103, 104; Volume 4: 425). A copper concentration of .02 
mg/1 would cause more than a 90% reduction in the ciliary beating of gills 
from large clams. The maximum zinc concentration (.1 mg/1) at the Mississippi 
sampling station was equal to or greater than the maxima at the four Illinois 
River stations (Illinois Environmental Protection Agency, 1975, Volume 2: 92, 
93, 103, 104; Volume 4: 425), and just within the range which caused a 90% 
reduction in the ciliary response of gills from large clams. 

A glance at Tables 2, 3, and 4 show that several toxic metals generally 
occur at higher concentrations in the Illinois River than in the Mississippi. 
In view of the extreme sensitivity of the clam gills to copper, lead, and zinc, 
it appears that metals in the Illinois River could be a significant stress on 
adult fingernail clams. This tentative conclusion, which is based on tests 
with the sensitive gill preparations, should be verified with bioassays using 
intact fingernail clams. 



46 



Response of Clams to Potassium Chloride 

Response of Gill Preparations . There was a marked difference in the 
response of gill preparations from small and large clams to potassium. When 

gills from small clams were exposed to a potassium concentration of 39 mg/1 

_3 
(10 M) , steady ciliary beating rates were maintained for eight days (basal 

rate). Greater concentrations (390 to 19,500 mg/1) caused the cilia beating 

rate to decline (Figure 14). Concentrations ranging from 3.9 to 0.0039 mg/1 



Q 

Z 

o 
o 
liJ 

(/) 

Q. 5 
(0 



UJ 



•T^ 




SMALL CLAMS 








r 


..^ 


lO'^M T 


(39 mg/l) 


T 


T 


■ u 


\ 


\. 




(390 mg/l) 






5xio-''m\ 

(19,500 mg/l)*^ 


V 

— i— - 


___T 


\^^^ Jio"''mt 


(3900 mg/l) 

2 


T 


^ 


' 1 1 


— 1 — 


1- 




, 







3 4 5 
DAYS 



Figure 14. Ciliary beating response of gills from small clams. A potassium 
concentration of 39 mg/l maintains a basal rate; higher concen- 
trations are cilio- inhibitory. 



caused a temporary increase in beating rate for three to four days, followed 
by a return to nearly the basal rate or slightly above (Figures 15 and 16). 
Lower concentrations ranging from 0.00039 to 0.000039 mg/l caused a greater 
increase in beating rate for one to four days, followed by a decline below 
the basal rate. Finally, the lowest concentration tested, 0.0000039 mg/l, 
failed to excite the cilia or to maintain the basal rate (Figure 15). 



47 




Figure 15. 



Ciliary beating response of gills from small clams. A potassium 
concentration of 39 mg/1 maintains a basal rate (dashed line) , 
while lower concentrations (3.9 and 0.039 mg/1) temporarily 
excite the cilia. 



30- 








SHALL CLAMS 




(.000039 

MG/L) 


1C 


'> 


10t ^ (.00039 mg/l) 
y^ (.0039 mg/l) 


Q 

Z 



20- 

UJ 

0) 

Qi 




/ 


't\/' 


10,'^M T 


/ 


f 


y 


\iOj^m\t \ 


0) 


/ 


/ 


'r- 


'SASsh'i mg/l)\j ^^T 



•-D- 



(.0000039 mg/l) 
— I 1— 



Figure 16. Ciliary beating response of gills from small clams. Continuous 

exposure of the gills to potassium concentrations of 0.039 and 0.0039 
mg/l causes an increase in ciliary beating rate for four days, 
followed by a return to nearly the basal rate (dashed li^i^) or 
slightly above. Potassium concentrations of 0.00039 and 0.000039 mg/l 
cause a greater increase in ciliary beating rate for one to three 
days, followed by a decline below the basal rate. The lowest con- 
centration, 0.0000039 mg/l, fails to maintain a basal rate. 



48 



The same pattern of response to potassium occurred in large clam gills 
as in small clam gills, but at much lower concentrations (Figures 17 and 18) 







CONCENTRATION OF K 






■^ 




T 




10-^M_^-^^' 




, ^^ 


■■\ 


V 






10_^^M (.39MG/L) 






-— 1 — 


— 1 — 


=i 


(39 mg/l) 


io' M r"*~— -~ 


^ 


^ 


h— 


" ! t - 





3 4 5 
DAYS 



Figure 17. Ciliary beating response of gills from large clams. A potassium 
concentration of 0.039 mg/1 maintains a basal rate; higher 
concentrations are cilio- inhibitory. 



Q 

Z 
O 10 

u 

U) 

q: 

OJ 

en 





r 10"®M (-000039 mg/l) 

T 

10"''°M '■0000039 mg/l) 



3 4 5 
DAYS 



Figure 18. Ciliary beating response of gills from large clams. Potassium 

concentrations of 0.0039 and 0.00039 mg/1 excite the cilia; while 
0.000039 and 0.0000039 fail to maintain the basal rate. 



49 



For example, basal ciliary beating rates in large clam gills were maintained 
in 0.039 mg/1 potassium (10~ M) . Higher concentrations (0.39 to 39 mg/1) 
caused cilio- inhibition, lower concentrations (0.00039 to 0.0039 mg/1) 
caused cilio-excitation, and still lower concentrations (0.0000039 to 
0.000039 mg/1) caused beating rates to decline almost to zero. 

Figure 19 shows that ciliary beating rates of gills from both large and 
small clams declined when the gills were exposed to molluscan saline solution 
containing no potassium for four days. When potassium was added to the 
solution, the gills from small clams showed a recovery pattern, while gills 
from large clams did not. 




Figure 19. Ciliary beating rate of gills from large clams fails to recover 
from inhibiting effect of potassium withdrawal. Gills from 
small clams do recover. 



50 



Gills from large and small clams were kept in molluscan saline solution 
containing the potassium level required for maintenance of basal ciliary 
beating rates, then exposed to a lower potassium level (0.00039 mg/1) which 
was cilio-excitatory for both. Figure 20 shows that gills from large clams 
lagged considerably behind gills from small clams in response to the potassium 
reduction. 



20 

Q 

z 
o 

U 15 

UJ 
U) 

a: 

(0 



- 










^^^ 


ttlh 




SMALL CLAMS ,JL ^ 




^r^^t^ 


- 




T 




Tii 


-L^-l 


nml 


*•»• 








v^ 




T*** 












•u 




1*^ 








T 


i4 


UVi;*^ 


1^ 


^LARGE 


CLAMS 






■L 


-LI 














i 

1 — h 


START 


IQ-S M (.00039 MG/L 
1 


K+ 




-1 




1 — 



1 2 

HOURS 



Figure 20. Gills from large clams show a greater lag than gills from small 
clams in response to a cilio-excitatory level of potassium. 



Acute Response of Intact Clams to Potassium Chloride . Death was the 
response used in the acute bioassays. The acute toxicity curve indicated 
that potassium was a slow-acting toxicant. Lethal thresholds or probable 
lethal thresholds developed between 260 and 400 hours (tests Jl, J2, and A2; 
Figures 21 and 22). In tests J3 and J4 (Figures 21 and 22), even after 600 
hours of exposure, lethal thresholds did not develop. The only test in which 



51 



>- 

5 iioo-i 

»— 



UJ 
Q- 






GO 



300- 



150- 

lOOH 
80 




=c - PROBABLE 

^ 60- LETHAL 
UJ THRESHOLD 

^ 50" 250 mg/ liter 



TEST Jl, JUVENILES 
I ^O^ 1 



I I ' I'M' 

I 300 I 600 I 1000 
200 ^00 800 



2000 



3000 



POTASSIUM CONCENTRATION mg/liter 



Figure 21. Comparison of acute toxicity curves, adult test Al and juvenile 
test Jl. The tests were conducted under the same conditions 
with 10 clams per concentration. The closed and open circles 
are concentrations which kill 50% of the clams (LC50) at dif- 
ferent observation times during the test. The 95 percent confidence 
limits of each LC50 are indicated. 



a valid lethal threshold developed, at a potassium concentration of 200 mg/1. 
was adult test A2 (Figure 22). Tests Jl (Figure 21) and J2 (Figure 22) 
developed probable lethal thresholds of 250 and 290 mg/1, respectively. 



52 



5 300 
Z 200 



° 100 






80 
60 



^0- 



LETHAL 
THRESHOLD 
200 mg/ liter 



TEST J2 JUVENILES 




H^t — I 

PROBABLE LETHAL THRESHOLD 290 mg/liter 



600 I 1000 

100 aoo 800 

POTASSIUM CONCENTRATION mg/liter 



2000 



3000 



Figure 22. Comparison of acute toxicity curves of adult test A2 and 

juvenile test J2. The tests were conducted under the same 
conditions with 20 clams per concentration. The open and 
closed circles are potassium LC50s at different observation 
times during the test. The 95 percent confidence limits for 
each LC50 are indicated. 



Toxicity curves of adult and juvenile clams under similar test condi- 
tions were compared (Figures 21 and 22) and were significantly different 
(p=0.05). Adults responded 5 to 1.6 times faster than juveniles. 



53 



Chronic Response of Intact Fingernail Clams to Potassium Chloride . Two 
chronic bloassays were conducted with potassium (Figures 23-26). In 
potassium bloassay Kl , the concentrations ranged from 11.9 to 195 mg/1 
potassium. There were no significant mortalities (Figure 23) or reductions 
in growth (Figure 24) after 42 days of continuous exposure to the highest 
concentration. The concentrations were increased in the second potassium 
bloassay K2 to produce a range from 14.3 to 275 mg/1 potassium. The only 



K mg/1 

-A = CONTROL 11.9 
= 115 




2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 
DAYS OF EXPOSURE 



Figure 23. In the first chronic bloassay with potassium (Kl), there 
were no significant differences in the mortality of clams 
exposed to the different potassium concentrations. Points 
which are not significantly different (p<.05) are included 
within a dashed-line box. 



54 



K (mg/1) 




^ _ _ n I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 

O" 2 ^ 6 8 10 12 m 16 is 20 22 2^ 26 28 30 32 34 35 38 40 42 



DAYS OF EXPOSURE 



Figure 24. In the first chronic bioassay with potassium (Kl) , there were 
no significant differences in the growth (in length) of clams 
exposed to the different potassium concentrations. Points 
which are not significantly different (p<.05) are included 
within a dashed-line box. 



concentration which produced significant mortalities after 14 days of 
exposure was the highest, 275 mg/1 (Figure 25). Thus the maximum 
acceptable toxicant concentration (MATC) for long-term survival of 
fingernail clams lies between 195 and 275 mg/1 potassium. Figures 24 and 26 
show the effect of potassium on the growth of fingernail clams during 
bioassays Kl and K2, respectively. The lengths of clams surviving the 
potassium concentration of 275 mg/1 (bioassay K2) were not used in the 



100 
90 - 
80 - 
70 - 
60 
50 
^0 
30 - 
20 - 
10 - 



K (mg/1 



55 



275 
14.3 
45 
184 



-3 

-O = 106 
-D = 65 




2 ^ 



1 — 1 — I ly^A I — I — r 

6 8 10 12 14 16 18 20 22 24 

DAYS OF EXPOSURE 



Figure 25. In the second chronic bioassay with potassium (K2) the mortality 
of clams exposed to 275 mg/1 was significantly greater (p<.05) 
than the mortality in the lower concentrations and in water 
with no added potassium. 



analysis of potassium effects on growth, since the reduced number of surviving 
clams would cause sample bias. In comparison with the controls, none of 
the potassium concentrations between 45 and 184 mg/1 caused reduction in 
growth. On the contrary, it appears that potassium actually stimulated the 
growth of the clams with maximum growth occurring at 106 mg/1. Potassium 
apparently has no sublethal effect on the growth of fingernail clams. The 
effects of potassium on the reproduction of the clams was not determined, 
because no reproduction occurred during these bioassays, even in the con- 
trols where no potassium was added. 



56 




T — I — I — I — I — I — I — I — I — I — r 

10 12 14 16 18 

DAYS OF EXPOSURE 



I — i—i — I — I — I — I — I 

20 22 24 26 28 



Figure 26. In the second chronic bioassay with potassium (K2) , there 

were no significant reductions in growth of the clams exposed 
to the different potassium concentrations. In fact, the 
potassium actually stimulated growth, with maximum growth 
occurring at a concentration of 106 mg/1. Points which are 
not significantly different (p<.05) are enclosed within a 
dashed-line box. 



The acute static bioassays indicate that the lethal threshold concen- 
tration (LC50) of potassium is 200 mg/1 for adult clams and probably 250- 
290 mg/1 for juvenile clams. The chronic bioassay data show that the maximum 
acceptable toxicant concentration (MATC) lies between 184 and 275 mg/1 K. 
In comparison to several other test organisms, M. transversum is sensitive 
to potassium. For example, the 96-hour LC50 for mosquite fish, Gambusia 
af finis , is 920 mg/1 potassium chloride (482 mg/1 potassium) (Wallen, Green, 
and Lasater, 1957) and for bluegill, Lepomis macrochirus , the 96-hour LC50 



57 



is 2010 mg/l potassium chloride (1054 mg/1 potassium) (Anonymous, 1960). In 
fish toxicology, the 96-hour LC50 is comparable to a lethal threshold. 

M. transversum was about as sensitive to potassium as several other 
invertebrates tested. The threshold concentrations for immobilization of 
Daphnia magna , Cyclops vernalis, and Mesocyclops leukarti are 430, 640, and 
566 mg/1 potassium chloride, respectively (Anderson et al. , 1948). These 
concentrations are equivalent to 225, 336, and 297 mg/1 potassium. The 
96-hour LC50 for the Asiatic clam, Corbicula manilensis , is 225 mg/1 potas- 
sium (Anderson et al. , 1976) which indicates that the Asiatic clam may be 
more sensitive to potassium than M. transversum . However, a lethal threshold 
was not determined. Imlay (1973) found that a potassium concentration of 
11 mg/1 was lethal to 90 percent of 3 species of unionid clams, Actinonaias 
carinata, Lampsilis radiata siliquoidea , and Fusconaia flava, in 36 to 52 
days of exposure; a potassium concentration of 7 mg/1 was fatal to the latter 
2 species in about 8 months. These species are considerably more sensitive 
to potassium than M. transversum . 

The highest potassium concentration in 25 water samples taken near the 
surface of the Illinois River in 1975 was 6 mg/1 potassium (Anderson, 1977), 
well below the lethal thresholds or probable lethal thresholds found in this 
study. However, before considering potassium levels in the Illinois River low 
enough to allow survival of the species , the microhabitat of the clam should 
be considered. Since the clams live in or on the sediments, they utilize 
water from the interface or interstitial water. Little is known concerning 
the chemistry of these waters in regards to equilibria with the water column 
and sediment. Considering that potassium concentrations as high as 250 mg/kg 
have been found in the sediments of the Illinois River by Mathis and Cummings 
(1971), it is very possible that water associated with these sediments provides 



58 



considerably higher potassium concentrations than water above the sediments. 
Additional research is needed in this area. 

The acute static bioassay results provided information important in 
population dynamics. The adults respond 5 to 1.6 times faster than juveniles 
tested under the same conditions and showed a significantly lower lethal 
threshold of 200 mg/1 potassium (Figure 22) when compared to the probable 
lethal thresholds of 250 and 290 mg/1 potassium (Figures 21 and 22, re- 
spectively) for the juveniles. This is important as the reproductive por- 
tion of the population would be affected first when stressed and this could 
mean that death would occur before the adults could reproduce. The larger 
the adults grow, and the longer they live, the more young they produce. 
Any reduction in the longevity or growth of adults therefore would decrease 
production of young. 

Effect of Water Hardness in Modifying Toxicity of Potassium Chloride . 
The effect of 2 different levels of water hardness on the response of the 
juvenile clams to potassium was determined (Figure 27). There was a 
significant difference (p<.05) in the response of the clams to potassium at 
the two levels of water hardness (Figure 27). Clams tested in the softer 
water, total hardness equal to 243 mg/1 as CaCO (test J2) , responded 
faster than clams tested in the harder water, total hardness equal to 314 
mg/1 as CaCO (test J3) , with the toxicity curve of the soft water test 
approaching the vertical asymptote at 400 hours (Figure 27). In contrast, 
the toxicity curve of the clams tested in the harder water does not show 
a lethal threshold even at 696 hours. 



59 




-*-TEST J3, TOTAL HARDNESS= 31^ 

mg/liter as CAC03 



I ' I ' r ' 

200 5OS0O 600 800^000 2000 3000 
POTASSIUM CONCENTRATION mg/liter 



Figure 27. Clams exposed to potassitim in hard water (314 mg/1 as CaCO ) 
did not die as rapidly as clams exposed to potassium in 
softer water (243 mg/1 as CaCO ) . 



Effect of Temperature in Modifying Toxicity of Potassium Chloride . The 
LC50s in the test where the water temperature was 6.5 C were significantly 
greater (p<.05) than in the test at 16.7 C, with the exception of the 96-hour 
LC50 (Figure 28). The toxicity curve of 16.7 C test approached the vertical 
asymptote in 400 hours whereas in the 6.5 C test a lethal threshold had not 
developed even after 648 hours (Figure 28) . It did appear that a vertical 
asymptote was being approached after 648 hours in the test at 6.5 C, and 



60 





800 




700 


>- 


600 


1— 




1 


bOO 


<x 




^ 


^00 


o 




ZI 




1 — 


300 






UJ 




t_> 




Od 




^ 


200 


C3 




in 


ISO 


c-> 




1— 




S 


100 






S 


80 






^ 


60 


1 — 


50 



TEST 



PROBABLE LETHAL 
THRESHOLD 
290 mg/ liter 



TEST J4. 6.5°C 




I 300 L ' _L'Jiobo 



200 



400 600 80( 



\ 3000 
2000 



POTASSIUM CONCENTRATION mg/liter 



Figure 28. Juvenile fingernail clams died more rapidly in warm water than 

in cool water when exposed to potassitim chloride, but the lethal 
thresholds at the two temperatures were probably almost the same. 



that the asymptotic value would have been close to that in the test at 16.7 C. 
The cold temperature apparently slowed the rate of uptake or the rate of 
toxic action of potassium, but did not change the lethal threshold . This 
means that an agent whose toxic action was similar to that of potassium 
would kill clams more slowly at cold water temperatures than at warm tempera- 
tures, but that if the exposure were continued long enough, the same per- 
centage of the population would be killed in both cases. 



61 



Response of Clams to Airanonivun Chloride # 

Response of Gill Preparations . Figure 29 shows that gills from large 
fingernail clams were more sensitive to un-ionized ammonia than gills from 
small clams. a 



SPHAERIUM 

TRANSVERSUM 

(SMALL) 

SPHAERIUM 

TRANSVERSUM 

(LARGE) 




NH3 (ppm) 



Figure 29. Ciliary beating response of gills from large and small fingernail 
clams to un-ionized ammonia (expressed as NH , ppm, or mg/1) . 

The un-ionized ammonia concentrations which induced various degrees of 
inhibition of the ciliary activity of gills from large and small clams are 
given below. The concentrations are expressed as un-ionized ammonia nitrogen . 
NH_-N mg/1, which is the way ammonia concentrations are reported by the 
Illinois Environmental Protection Agency. The un-ionized ammonia values in 



Figure 29 were converted to NH_-N by multiplying by .824. 



62 



Gills from 
large clams 

Gills from 
small clams 



50% reduction 
in ciliary 
beating rate 

.03 mg/1 
.06-. 07 mg/1 



90% reduction 
in ciliary 
beating rate 

.04 mg/1 



,08 mg/1 



complete ''' 
inhibition 
of cilia 

.05-. 06 mg/1 
.08-. 09 mg/1 



Adult fingernail clams are slightly more sensitive to un-ionized ammonia 
than the Asiatic clam ( Corbicula manilensis ), a freshwater unionid mussel 
( Elliptio complanata ) , and a marine mussel ( Mytilus edulis ) , as can be seen 
by comparing Figures 29 and 30. 



T'' = 18°C 
PH = 7.5 
(Oj) 8ppm 




NH3 (ppm) 



Figure 30. Ciliary beating response of gills from the Asiatic clam ( Corbicula 

manilensis ) , a freshwater mussel ( Elliptio complanata ) , and a marine 
mussel (Mytilus edulis ) , to un-ionized ammonia (expressed as NH , 
ppm, or mg/1). Note .11 on horizontal scale should be .10. 



63 



The dose-response curves for large and small fingernail clams display a 
classic sigmoid pattern (Figure 29), while the decrease in the ciliary response 
of the Asiatic clam is so abrupt between .05 and .06 mg/1 NH that the curve 
is almost rectilinear (Figure 30) . The ciliary response of Elliptio complanata 
decreases linearly with increasing concentration of un-ionized ammonia. The 
un-ionized ammonia concentrations which produced various levels of ciliary 
inhibition in the three species, other than fingernail clams, are given be- 
low (concentrations are expressed as un-ionized ammonia nitrogen, NH -N, mg/1): 

50% reduction 90% reduction complete 
in ciliary beating in ciliary beating inhibition 
rate rate of cilia 

Elliptio complanata .06 mg/1 .09 mg/1 .09-. 10 mg/1 

Mytilus edulis .08 mg/1 .09 mg/1 .09-. 10 mg/1 

Corbicula manilensis .05 mg/1 .06 mg/1 .09-. 10 mg/1 

Effect of Oxygen in Modifying Toxicity of Ammonium Chloride . Figure 31 
shows that increasing the dissolved oxygen concentration in the water above 
the saturation concentration of 9.18 mg/1, reduces the inhibitory action of 
un-ionized ammonia on the cilia of the gills. 

At the same NH -N concentration of .07 mg/1, the ciliary beating rate of 
gills from large clams was barely maintained at the highest oxygen concen- 
tration tested, 16 mg/1 (170% of saturation). Figure 32. 

These results indicate that the sensitivity of the gill preparations to 
un-ionized ammonia increases as the oxygen content of the water decreases. 



64 



14-r 



SPHAERIUM TRANSVERSUM (SMALL) 

(16 ppm.Og) 



0.07 NH3-N (ppm) 

T°=18°C 

PH = 7.5 



(14 ppm.Og) 




15 20 25 
(MIN.) 



30 



Figure 31. Ciliary beating rates of gills from small clams did not decrease 

when the gills were exposed to .07 mg/1 NH^-N and oxygen concentra- 
tions of 14 mg/1 or more (150% or more of the oxygen saturation 
concentration of 9.18 mg/1). 

SPHAERIUM TRANSVERSUM (LARGE) 



12-1- ^-^"^ NH3-N(ppm) T°=18°C PH = 7.5 



^ ,-^^(16 ppm.02) 
(14 ppm.Og) 




Figure 32. The ciliary beating rates of gills from large fingernail clams 

decreased markedly when the gills were exposed to .07 mg/1 NH»-N, 
and oxygen concentrations below 12 mg/1 (130% of saturation). Even 
at the highest oxygen concentrations of 14 and 16 mg/1 (150% and 
170% of saturation) the ciliary beating rates declined slightly. 



65 



Chronic Response of Intact Clams to Ammonium Chloride . Two chronic 
bioassays were conducted with ammonia (Figures 33-36). In chronic bioassay NH 2 
significant mortalities occurred in the upper 2 ammonia concentrations, 
0.59 and 0.93 mg/1 undissociated NH -N, after 42 days of continuous exposure 
to ammonia (Figure 33) . This results in a maximum acceptable toxicant con- 
centration (MATC), based on mortality, between 0.35 and 0.59 mg/1 un-ionized 
NH -N. Growth data from the test concentrations where there was not a 
significant difference in mortality indicate that there was no effect on the 



100 

90 

80 
70 
60 
50 
i*0 
30 
20 
10 




= 0.93(0.46-1.53) 
= 0.59(0.34-0.82) 



■T^ = CONTROL 0.01(0.004-0.01; 

-O = 0.35(0.17-0.49) 

-• = 0.10(0.05-0.13) 

-a = 0.20(0.11-0.27) 



r-n 




2 4 6 8 10 12 14 16 18 20 22 24 25 28 30 32 34 36 38 40 42 
DAYS OF EXPOSURE 



Figure 33. Un-ionized ammonia concentrations of .93 and .59 mg/1 (as NH^-N) 
caused significant (p<.05) mortality among fingernail clams after 
42 days of exposure. All points within the same box are not 
significantly different (p<.05). 



66 



growth, e.g. growth was not reduced below that of the control (Figure 34). 
However, growth was not good during the experiments with a maximum growth 
of only 0.5 mm in 42 days. It was suspected that the slow growth was due 
to the small size of clams that were used at the initiation of the experi- 
ment (2.1-2.3 mm) and the possibility that the clams were born in the 
laboratory under stress conditions. Subsequent work showed that using 
clams that averaged 2.5 mm or greater in length resulted in better growth. 



2.6 -I 



IE 

t£3 



-D = 0.20(0.11-0.27) 

-• = 0.10(0.05-0.13) 

-A = CONTROL 0.01(0.004-0.01) 

-O = 0.35(0.17-0.49) 

— = 0.59(0.34-0.82) 

•- = 0.93(0.46-1.53] 




"» 1 — I — I 1 1 1 — I — i 1 — I 1 1 1 — I 1 — I — I 1 — r 

^2 4 6 8 10 12 li| 16 18 20 22 2A 26 28 30 32 5^ 36 38 40 42 

DAYS OF EXPOSURE 



Figure 34. Results of chronic bioassay NH 2. Un-ionized ammonia concentrations 
of 0.10 and 0.20 mg/1 actually stimulated the growth of fingernail 
clams, presumably by stimulating the growth of bacteria upon which 
the clams feed. Growth at all other concentrations was not sig- 
nificantly different (p<.05) from that in the control. 



67 



A second ammonia chronic bioassay, NH 3, was conducted in an attempt to 
obtain adequate growth data to estimate the MATC for growth. The upper con- 
centration of un-ionized ammonia (1.20 mg/1 NH -N) caused significant mor- 
tality after 14 days of exposure, and the next highest concentration (0.60 mg/1 
NH--N) caused significant mortality after 42 days (Figure 35) . Thus the 
MATC, based on mortality, lies between 0.34 and 0.60 mg/1 un-ionized ammonia 
(NH_-N) . These results confirmed the results obtained in test ^^2^' 




14 



18 22 26 
DAYS OF EXPOSURE 



30 5^ 38 42 



Figure 35. An un-ionized ammonia concentration of 1.20 mg/1 caused 

significant (p<.05) mortality among fingernail clams after 
14 days of exposure, and a concentration of 0.60 mg/1 caused 
significant mortality after 42 days. Mortalities at the other 
concentrations were not significantly different from controls. 



68 



Clams in un-ionized ammonia concentrations where significant mortality 
occurred were not used to determine effects of ammonia on growth, because 
the sample might become biased if the different size classes of clams dif- 
fered in their susceptibility to ammonia. For example, the experiments with 
the gill preparations demonstrated that gills from large clams were more 
sensitive to ammonia than gills from small clams. If large clams are more 
readily killed by ammonia than small clams, then the average size of clams 
in lethal concentrations of ammonia would diminish during the course of an 



NH,-N(mg/1) 



^.0- 

3.! 
3.6- 

3.2H 



= 0.10(0.07-0.14 

= 0.20(0.14-0.27) 
-A = CONTROL 0.01(0.003-0.01) 
— = 0.60(0.43-0.74) 
■- = 1.20(0.84-1.53) 
-O = 0.34(0.27-0.46) 




4-1' I ' I ' I ' I ' I ' I ' I ■ I ■ I ■ I 

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 

DAYS OF EXPOSURE 



Figure 36. Un-ionized ammonia concentrations of 0.34 and 0.60 mg/1 (NH -N) 

significantly (p<.05) reduced the growth of fingernail clams after 
14 days of exposure. Growth of clams was significantly enhanced 
at lower concentrations probably due to increased growth of 
bacteria upon which the clams feed. All data points within the 
same box are not significantly different (p<.05). 



69 



experiment, in comparison to clams in sublethal concentrations, but the effect 
would be attributable to size-selective mortality, rather than to a direct 
effect of ammonia on growth. ■ 

Figure 36 shows that un-ionized ammonia concentrations of 0.34 and 0.60 
mg/1 (NH -N) significantly (p<.05) reduced the growth of fingernail clams 
after 14 days of exposure. The MATC, based on growth, lies between 0.20 and 
0.34 mg/1 un-ionized ammonia (NH„-N) . 

The results of the chronic bioassays with ammonia are summarized below: 

Test No. MATC based on mortality MATC based on growth 

NH^2 >0.35<0.59 mg/1 NH^-N 

NH 3 >0.34<0.60 mg/1 NH„-N >0.20<0.34 mg/1 NH^-N 

Effect of Sublethal Exposure to Ammonium Chloride on Subsequent Response 
to Stress . Fingernail clams which had been exposed for 44 days to well water 
containing no added airanonia or to sublethal concentrations of 0.10 and 0.20 
mg/1 un-ionized ammonia in test NH„3 were delivered to Southern Illinois 
University for testing in the cilia monitoring apparatus. They were held 
for one week in a culture tank containing no added ammonia before their gills 
were removed and tested. 

Figure 37 shows that chronic exposure of clams to sublethal concentrations 
of ammonia alters the response of their gills to potassium. The ciliary 
beating rate of gills from clams previously exposed to 0.1 mg/1 NH_-N was sub- 
sequently stimulated at potassium concentrations of 10 to 10 M (3. 9-. 39 mg/1) 
whereas gills from clams not exposed to ammonia showed maximum stimulation 
at potassium concentrations ranging from 10~ to 10~ M (. 39-. 0000039 mg/1). 
The maximum ciliary beating rate of the gills exposed to 0.1 mg/1 NH„-N was 
only 15, whereas the maximum was 28 for the unexposed group. Previous exposure 



70 



MUSCULIUM TRANSVERSUM (SMALL! 




Animals not exposed to NH3 
O-Q Animals exposed to 01 mg/l NH3 (or 44 days prior to K 1 
A A Animals exposed to 0.2 mg/l NH3 for 44 days prior to K I 



Figure 37. The ciliary beating response to potassium of gills from clams 

previously exposed for 44 days to sublethal NH„-N concentrations 
of 0.1 and 0.2 mg/l was markedly altered in comparison to the 
response of gills from clams not previously exposed to ammonia. 



to 0.2 mg/l NH„-N completely blocked the stimulatory response to potassium 
addition. In fact, the ciliary beating rate was slightly to markedly in- 
hibited at all potassium concentrations. 



71 



MUSCULIUM TRANSVERSUM (SMALL) 



T°= 18 C 
pH = 7.5 
©2 = 8 ppm 



Animals not exposed to NH3 
O-O Animals exposed to 0.1 mg/l NH3 for 44 days prior to NH3 testing 
A— A Animals exposed to 0.2 mg/l NH3 for 44 days prior to NH3 testing 




NH3 - N (ppm) 

Figure 38. When clams are exposed to sublethal concentrations of un-ionized 
ammonia (0. 1 and 0.2 mg/l NH -N) for 44 days, their gills are 
sensitized to subsequent additions of ammonia. 



Figure 38 shows that the gills of the clams are sensitized to ammonia 
by previous exposure to sublethal concentrations of aimnonia. A 50% reduction 
in the ciliary beating rate occurs at an un-ionized ammonia concentration 
of .09 mg/l (NH -N) in control gills not previously exposed to ammonia, at 
.04 mg/l in gills previously exposed to 0.1 mg/l NH„-N, and at .01 to .02 
mg/l in gills previously exposed to 0.2 mg/l NH„-N. 



72 



MUSCULIUIV TRANSVERSUM (SMALL) 



pH = 7.5 

NH3 ■ N = .01 ppm 

O2 = 8 ppm 



9-9 Animals not exposed to NH3 

00 Animals exposed to 0.1 mg/l NH3 for 44 days prior to T° testing 

A_A Animals exposed to 0.2 mg/l NH3 for 44 days prior to T° testing 




TEMPERATURE ("C) 



Figure 39. Previous exposure of clams to sublethal concentrations of 
un-ionized ammonia reduces the maximum ciliary response of 
their gills to temperature and also reduces the temperature 
range in which normal ciliary activity can be maintained. 



Figure 39 shows that prior exposure to ammonia reduces both the maximum 
ciliary response to temperature and the temperature tolerance range. While 
previous exposure to 0.1 mg/l NH^-N has a rather small, but detectable 
effect on the temperature response, previous exposure to 0.2 mg/l has a 
dramatic effect. 



73 



MUSCULIUM TRANSVERSUM (SMALL) 




Animals not exposed to NH3 
00 Animals exposed to 0.1 mg/l NH3 for 44 days prior to Oj testing 
^_^ Animals exposed to 0.2 mg/l NH3 for 44 days prior to O2 testing 



CONCENTRATION OF Oj (ppni) 



Figure 40. Previous exposure of fingernail clams to sublethal concentrations 
of un-ionized ammonia reduces the ability of the gills to 
increase their ciliary beating rate in response to increasing 
concentrations of oxygen. 



Figure 40 shows that previous exposure of fingernail clams to 
sublethal concentrations of un-ionized ammonia reduces the ability of the 
gills to increase their ciliary beating rate in response to increasing 
concentrations of oxygen and Figure 41 shows that decreasing concentrations 
of oxygen have a greater inhibitory effect on the ammonia-exposed gills 
than on the gills not exposed to ammonia. 



74 



MUSCULIUM TRAIMSVERSUM (SMALL) 



T = 18 C 
pH = 7.5 
NH3 - N = .01 ppm 



Animals not exposed to NH- 



0—0 Animals exposed to 0.1 mg/l NH3 for 44 days prior to O2 testing 
A A Animals exposed to 0.2 mg/l NH3 for 44 days prior to O2 testing 




CONCENTRATION OF O2 (ppm) 

Figure 41. Low oxygen concentrations cause greater inhibition of ciliary 
beating rates on gills from clams previously exposed to 
sublethal concentrations of ammonia than on gills from clams 
not exposed to ammonia. 



Relative Sensitivity of Fingernail Clams and Other Aquatic Organisms to 
Ammonia . The results of the chronic ammonia bioassay indicate that M. trans- 
versum is sensitive to un-ionized ammonia. Data from ammonia bioassay NH 2 
demonstrated a MATC for survival between 0.35 and 0.59 mg/l un-ionized 
NH -N. Data from ammonia bioassay NH.,3 demonstrated a MATC for survival be- 
tween 0.34 and 0.60 mg/l un-ionized NH -N and a MATC for growth between 
0.20-0.34 mg/l un-ionized NH„-N. The sensitivity of M. trans versum to 
ammonia is similar to the sensitivity of rainbow trout with concentrations 



75 



between 0.4 and 1.8 mg/1 un-ionized airanonia reported to be lethal to rainbow 
trout depending on the free CO^ and dissolved oxygen concentrations (Merkens 
and Downing, 1957, Lloyd and Herbert, 1960). The bluegill sunfish, Lepomis 
macrochirus , a warm-water species, is more tolerant of ammonia than M. 
transversum with the lethal threshold reported as 1.65 mg/1 un-ionized NH^ 
(1.36 mg/1 NH^-N) (Lubinski, Sparks, and Jahn, 1974). The only usable 
ammonia data for an invertebrate species that could be found in the litera- 
ture was a 2-day LC50 of 0.66 mg/1 un-ionized NH (0.54 mg/1 NH^-N) for 
Daphnia magna (European Inland Fisheries Advisory Commission, 1970). 

Although no data are available yet, it is probable that the reproduction 
of the fingernail clam could be affected at un-ionized ammonia concentrations 
below 0.20-0.34 mg/1 (NH -N) . The ciliary response of gills from large clams 
is markedly affected at concentrations of .03 mg/1 NH„-N. The ciliary beating 
response of adult fingernail clams is slightly more sensitive to un-ionized 
ammonia than that of the Asiatic clam ( Corbicula manilensis ) , a freshwater 
unionid mussel ( Elliptio complanata ) , and a marine mussel ( Mytilus edulis) , 
as can be seen by comparing Figures 29 and 30. Chronic exposure of clams to 
sublethal concentrations of ammonia lowers the tolerance of the gills to a 
variety of stresses, including additional exposure to ammonia (see discussion 
in the above section) . 

Relationship between Ammonia Levels in the Illinois and Mississippi Rivers 
and Ammonia Levels Which Affected Fingernail Clams in Laboratory Experiments . 
The total NH -N concentrations in the Illinois and Mississippi Rivers in 1975 
(Tables 2-4) were converted to un-ionized ammonia concentrations (NH -N, mg/1) 
using tables provided by Thurston et al. (1974: 9-15), and mean or median pH 
and temperature values reported by the Illinois Environmental Protection Agency 



.012 


.031 


.012 


.021 


.027 


.018 


.074 


.098 


.048 



76 



(1975, Volume 4: 425; Volume 2: 92-93, 103, 104). The median pH and temperature 
were used to convert the median ammonia concentrations (Table 2) , while the 
mean pH and temperature were used to convert both the mean and the maximum 
ammonia concentrations (Tables 3 and 4) . The un-ionized ammonia nitrogen 
concentrations (NH„-N, mg/1) occurring in 1975 are given below: 

Mississippi River Illinois River 

Rt. 9 Bridge Rt. 150 Bridge Lock and Dam Rt. 9 Bridge Rt . 97 Bridge 
Ft. Madison, lA Peoria, XL Creve Coeur, XL Pekin, XL Havana, XL 
Mile 383.9 Mile 165.8 Mile 157.7 Mile 152.9 Mile 119.5 

Median .005 .021 

Mean .010 .027 

Maximum .058 .125 

Although it would be better to convert the total ammonia concentrations to 
un-ionized ammonia concentrations using the pH and temperatures actually oc- 
curring at the time the ammonia samples were taken, rather than mean or 
median pH and temperatures, the results do indicate that the concentrations 
of un-ionized ammonia in the Illinois River in 1975 were approximately twice 
the concentrations in the Keokuk Pool, Mississippi River. Moreover, the mean 
and median concentrations at the four Illinois River stations where fingernail 
clams have died out were close to the value of .03 mg/1 which caused a 50% 
reduction in the ciliary beating rate of gills from large fingernail clams. 

Recently, Thompson and Sparks (1977) reported an alarming decrease in 
the fingernail clam populations in the Keokuk Pool, Mississippi River 
(Figure 42). 

Not only did the number of clams decline, but the growth of the sur- 
vivors was reduced (Figure 43). Xn 1976, the maximum shell length was only 



77 



NUMBER OF FINGERNAIL CLAMS PER M^ IN KEOKUK POOL. 
MISSISSIPPI RIVER. RIVER NILE 376.5. 1973-1977. 
SHADED AREAS INDICATE PERIODS OF DIVING DUCK 
UTILIZATION DURING SPRING AND FALL 
WATERFOWL MIGRATIONS. 




J S 

A 
-1973 



N J|M 

DFAJAO DFAJ AODF AJAO DFAJ AOD 
J 197^ ^ 1975 ^ 1976 ^ 1977 - 



Figure 42. The peak number of fingernail clams in bottom samples from 
Keokuk Pool, Mississippi River declined by 90% in 1976-1977. 



8.3 mm as compared to 12.4 mm in both 1974 and 1975. Fingernail clams 
begin to reproduce when they reach about 5 mm in shell length (Gale, 1969) 
In 1974 and 1975, the reproductive population numbered 5,000-6,000 clams 
per square meter. In 1976, the reproductive population was reduced to 
less than 1,000 per square meter. 



78 



FINGERNAIL CLAM SIZE CLASS 
DISTRIBUTION IN NOVEMBER 
OF 1974. 1975. AND 1976 
IN KEOKUK POOL. MISS. RIVER 




100 

10 



100 

10 

1 

.10 

.01 



il975 




-L 1976 



I I I I I I I 



-P»tncncr»a^ -^ coco vn^^o 






00 -t. o o^ 



CLAM SIZE CLASSES. SHELL LENGTH, mm 



Figure 43. The growth of fingernail clams in the Keokuk Pool, Mississippi 
River, was reduced in 1976. In 1976 the maximum individual 
shell length was only 8.3 as compared to 12.4 in both 1974 
and 1975. 



79 



MISSISSIPPI RIVER DISCHARGE (m^/second) 

AT KEOKUK. IOWA. 1973-1977 (monthly averages) 



5 - 



i< - 



t^ 3 - 




I I III I I I 11 l lll I I 1 1 11 11 Mil III 11 11 11 11 MM Mil I 11 11 II I 

A p p F A J AO p F A J Ap|D F a|j |a |o|D | F|A | J|A | 0|D 

J SNJMMJ S NJMM JSNJ MMJ S NJMMJ SN 



1973- 



•197^1 



1975' 



■1976- 



1977- 



Figure 44. Mississippi River discharge at Keokuk, Iowa, showing the effects 
of the 1976-1977 drought. 



Both the low clam population and reduced growth Thompson and Sparks ob- 
served in 1976 and 1977 appeared to be related to an extremely low river dis- 
charge as a consequence of a drought in the upper Mississippi Basin. In 
Figure 44, the Mississippi discharge at Keokuk, Iowa is graphed. The graph 
shows the usual cycle of spring highs and summer lows. However, the low dis- 
charge period in 1976 and spring discharge in 1977 were much reduced from those 
of previous years. During this low discharge period, Thompson and Sparks found 
effects on certain water parameters such as lower dissolved oxygen concentra- 
tions, dissolved oxygen stratification, increased water clarity, and the 
elevation in concentrations of certain materials such as un-ionized ammonia 



(NH^-N) . 



80 



NH3(u)-N CONCENTRATIONS IN KEOKUK POOL 

MISSISSIPPI RIVER. JULY-DECEMBERa973 & 1976 
WEEKLY MAXIMUM VALUES CALCULATED FROM TOTAL NH3- 

DATA PROVIDED BY CHEVRON (ORTHO) CHEMICAL CO.. 
FORT MADISON. IOWA il^-HOm COMPOSITE 
INTAKE SAMPLES) 




I I I I I I I I I I I I I I I I I I I I I I I 

JULY AUG. SEPT. OCT. NOV. DEC. 



Figure 45. The concentration of un-ionized ammonia (NH3-N) in the Keokuk 
Pool, Mississippi River, was greater in 1976 than in 1973. 



Figure 45 compares the weekly maximum concentrations for un-ionized 
ammonia for the low discharge period July-December in 1973 and 1976. We 
found that during the 1976 period that these values were elevated well above 
the 1973 concentrations. The highest value of 0.198 mg/1 NH -N is very 
near the level of 0.34 mg/1 which affected the growth of fingernail clams in 
our laboratory bioassays after two weeks of continuous exposure, and the 
average values of .02 to .03 mg/1 were close to, or within the range which 
caused a 50% reduction in the ciliary beating rate of gills from large clams. 

Our results with the gill preparations show that they become more sen- 
sitive to low oxygen levels and ammonia, following chronic exposure to 



81 



un-ionized ammonia concentrations of 0.10 or 0.20 mg/1. Since the dissolved 
oxygen levels on the bottom in Keokuk Pool were reduced during the drought, 
and toxicants, such as heavy metals, were presumably not diluted as much in 
1976-1977 as in previous years, one can speculate that the combined action 
of all these factors was sufficient to stress the fingernail clams in Keokuk 
Pool. Our results also showed that as the fingernail clams grow, they 
become less tolerant of extremes in environmental factors such as tempera- 
ture and low dissolved oxygen, and less tolerant of toxicants. 

We speculate that in 1976-1977, the fingernail clams in Keokuk Pool grew 
up to the point where their ciliary activity was impaired by environmental 
factors. At that point, their ability to feed and respire would be impaired, 
and they would grow slowly, if at all. Since the clams must reach a size of 
approximately 5 mm to reproduce, and the number of young produced increases 
as the size of the parent increases, the severe reduction in growth also 
caused a severe reduction in reproduction, hence reduced numbers of clams in 
1976-1977. This interpretation could be confirmed by reproducing the 1976- 
1977 conditions in the laboratory, and observing the effect on clam growth 
and reproduction. 

Response of Clams to Suspended Particles 

Table 6 shows that the particle transport rate of gills from large 
clams was more sensitive to suspended particles than the transport rate of 
gills from small clams. Table 6 also shows that in solutions containing 
equal numbers of particles per liter, sharp silica particles impaired the 
transport rate more than rounded illite clay particles. The 
transport rate was reduced more at low concentrations of oxygen when the 
particle concentration was increased. The particle concentrations (number 



82 



Table 6 . Effect of Oxygen Tension and Suspended Particles on the Average 
of Transport of Particles on the Gills of Musculium transversum.^ 



Rate 



Particles 






Averag 


e Rate 


of Transp( 


art 


(y 


m/i 


sec.-l 


) 




Per Liter 




Small Clams 








L 


ari 


ge Clams 






2 ppm 


4 ppm 


6 ppm 


8 ppm 


10 ppm 


2 ppm 


4 PI 


3m 6 ppm 8 ppm 
idard deviation = 


10 ppm 
0.9 ym) 


(control) 


Illite 


Clay Particles (Mear 


L size = 


2.8ym, 


stai 


155. 3^ 
+12.2 


150.2 


168.9 
+ 11.8 


169.7 
+ 10.2 


165.4 
+ 2.8 


10, 
+0. 


.1 
.3 


28, 
+0, 


.1 
.7 


40.0 
+0.9 


80.1 
+0.5 


90.3 
+6.9 


5xlo' 


30.2 
+13.8 


28.7 
+1.2 


89,2 
+ 10.2 


180.0 
+10.3 


151.2 
+10.2 


8. 
+0, 


,3 
,3 


11. 
+0. 


,3 
.1 


30.1 
+0.4 


60.2 
+0.1 


80.5 
+7.8 


io2 


12.2 
+12.8 


15.2 
+0.8 


70.2 
+9.7 


153.4 
+ 5.8 


161.3 
+ 1.3 


3. 
+0. 


.0 

,1 


3. 
+0. 


.3 
,1 


11.3 
+0.2 


48.2 
+0.2 


72.8 
+3.2 


103 


10.2 
+0.8 


11.2 
+0.1 


50.0 
+2.8 


138.3 
+ 3.2 


141.3 
+21.2 












7.3 
+0.5 


31.4 
+0.6 


61.3 
+ 1.9 


10* 


10.3 
+0.1 


10.3 
+0.3 


42.0 
±0.-8 


110.3 
+ 3.9 


128.4 
+ 8.2 












3.3 
+0-3 


15.3 
+0.9 


52.9 
+ 1.2 


10= 


10.2 
+0.1 


7.3 
+0.6 


43.3 
+0.8 


90.8 
+3.2 


115.1 
+ 12.2 












1.6 
+0.2 


12.3 
+0.3 


20.4 
+0.5 


10« 


10.2 
+0.1 


8.5 
+0.6 


38.2 
+0.2 


60.2 
+1.9 


110.1 
+ 8.2 















10.7 
+0.1 


21.3 
+0.7 


lo' 


9.8 
+0.2 


10.8 
+0.7 


27.5 
+1.6 


31.2 
+3.9 


61.2 
+9.8 















3.2 
+0.1 


19.8 
+0.2 


(control) 


Silica 


Flour 


Particl. 


28 (Mean size = 


3.3 


ym. 


standard deviation = 


= 0.4 ym) 


32.3 
+2.3 


50.2 
+ 1.8 


149.4 
+17.7 


143.7 
+ 3.2 


155.8 
+ 9.6 


9. 
+0. 


5 
8 


15. 
+ 1. 


5 
2 


30.2 
+3.9 


39.6 
+ 1.8 


55.2 
+10.2 


5x10^ 


17.2 
+0.8 


28.1 
+2.3 


32.3 
+2.8 


58.7 
+8.9 


109.2 
+ 7.8 







2. 
+0. 


3 
8 


15.3 
+3.2 


18.7 
+ 1.8 


25.2 
+8.9 


10^ 


2.1 
+0.6 


15.2 
+3.5 


21.8 
+3.5 


42.6 
+8.6 


92.6 
+6.9 















11.2 
+1.9 


15.2 
+6.3 


103 








9.3 
+1.7 


31.2 
+2.5 


91.9 
+9.3 















8,5 
+1.2 


6.2 
+0.8 


10* 








6.5 
+0.8 


12.8 
+0.8 


82.7 
+11.2 















6.3 
+0.8 


1.2 
+0.1 


10= 








3.2 
+0.2 


2.3 
+0.2 


61.2 
+6.3 


















1.8 
+0.1 


10^ 








3.1 
+0.2 


1.8 
+0.1 


42.7 
+2.5 





















lo' 














35.8 
+1.9 






















^Each point represents an average of 14 gills and 
a total of 168 observations. 



12 



readings per gill, or 



Mean + standard deviation. 



83 



per liter) which caused £t least a 50% reduction in the particle transport rates 

of gills from large and small clams at various oxygen concentrations, are given 

below. The weight of suspended matter per liter, in mg/1, is also given. 

Where one concentration caused less than a 50% reduction and the next higher 

concentration caused more than a 50% reduction, a range is given to indicate 

that the concentration having a 50% effect lay between the two concentrations 

which were tested. The numbers of particles per liter were converted to 

milligrams per liter using the following relationships: 2.3 x 10 illite 

particles per liter weighed 233.8 mg/1, and 3.6 x 10 silica particles per 

liter weighed 138.2 mg/1. 

Concentrations of Suspended Particles, or Concentration Ranges, Causing At Least 
a 50% Reduction in Particle Transport Rates of Clara Gills 

Illite Clay Particles 

Small Clams 
10^ - 10^ particles/1 



D.O., mg/1 
10 

8 
6 
4 
2 

10 
8 
6 
4 
2 



102 - 1,016 mg/1 

10 particles/1 
10.2 mg/1 

5 X 10 particles/1 
.005 mg/1 

10^ - 5 X 10^ particles/1 
.001 - .005 mg/1 

10^ - 5 X 10^ particles/1 
.001 - .005 mg/1 

Silica Flour Particles 

10 particles/1 
.384 mg/1 

5 X 10 particles/1 
.002 mg/1 

10^ - 5 X 10^ particles/1 
.0004 - .002 mg/1 

5 X 10 particles/1 
.002 mg/1 

5 X 10 particles/1 
.0004 mg/1 



10^ - 10^ particles/1 



Large Clams 

10 particles/1 

1.02 mg/1 

2 3 
LO - 10^ 

.01-. 10 mg/1 

5 X 10^ - 10^ particles/1 
.005 - .01 mg/1 

5 X 10 particles/1 
.005 mg/1 

5 X 10^ - 10^ particles/1 
.005 - .01 mg/1 



5 X 10 particles/1 
.002 mg/1 

5 X 10 particles/1 
.002 mg/1 

5 X 10^ particles/1 
.002 mg/1 

.0004 - .002 mg/1 



5 X 10 particles/1 



10' 



5 X 10 particles/1 



,0004 - .002 mg/1 



84 



Total suspended solids (TSS) was measured by the Illinois Environmental 
Protection Agency just once in 1975 at one of the four sampling stations in 
the reach of the Illinois River where the fingernail clams died out, and TSS 
was not measured at all at the station on the Keokuk Pool, Mississippi River. 
The one TSS value for the Illinois River at the U.S. 150 bridge at Peoria was 
700 mg/1. The suspended sediment in the Illinois River probably is comprised 
largely of illite clay, and 700 mg/1 thus would be sufficient to reduce the 
particle transport rate of gills from small clams by at least 50%, even if the 
oxygen levels in the water were at saturation or above. If the oxygen levels 
in the water were lower, the reduction in particle transport rates should be even 
greater, and gills from large clams would suffer even greater transport inhibition 
than gills from small clams. 

Response of Clams to Raw Illinois River Water 

Response of Gill Preparations . Figure 46 shows that the beating of 
cilia on the gills of small fingernail clams was almost completely inhibited 
when the gills were exposed to water taken from the Illinois River on 
October 5, 1977. Normal ciliary activity was maintained vzhen clam gills 
were exposed to water taken from a shallow, sand-point well located 
100 feet from the Illinois River. Partial inhibition of the cilia 
occurred when the river water was diluted with the well water. No additional 
effect on the cilia was observed after the diluted water had been stored in 
a metal reservoir, coated with aluminum paint, for several days. 



85 



MUSCULIUM TRANSVERSUM (SMALL) 



T = 18°C 
pH = 7.5 
O2 = 8 ppm 




WELL 



RIVER-WELL (32:68) 



RESERVOIR 



RIVER 



(HOURS) 



Figure 46. The ciliary activity of gills from small fingernail clams 

was almost completely inhibited when the gills were exposed 
to water taken from the Illinois River. Normal ciliary 
activity was maintained in well water. An intermediate 
response occurred in river water diluted with well water 
and no additional effect on the gills was obtained after 
the diluted water was stored several days in an aluminum- 
painted steel reservoir. 



86 




Figure 47. The white arrows show 

the curved shells which 
developed when the clams 
were exposed to Illinois 
River water which had 
been contaminated with 
metals. The shells were 
curved back to such an 
extent that the clam could 
not completely close them. 
All the clams with de- 
formed shells eventually 
died. 



Chronic Response of Clams to Raw Illinois River Water . On September 7, 
1977 fingernail clams were collected from the Keokuk Pool and delivered to 
the laboratory at Havana, where they were acclimated to well water for one 
month. Starting October 12, 1977 the clams were exposed to continuous flows 
of raw Illinois River water, well water, or to river water diluted with well 
water. During the next four weeks, the clams developed shell deformities 
(Figure 47) and died without reproducing, with the greatest mortality and 
highest incidence of shell deformities appearing in test chambers containing 
the most river water. 

Unfortunately, subsequent analysis of the river water revealed that the 
water had become contaminated with metals while it was being pumped from the 



87 



river into the test chambers. The metal content of the water in the river, 
and of the same batch of water after it had been pumped through the reservoirs 
and diluters, is presented below, for comparison. 



Concentration in 
river water (mg/1) 



potassium 


8.3 


aluminum 


2.1 


calcium 


58 


cadmium 


<0.01 


chromium 


<0.01 


cobalt 


<0.01 


copper 


0.23 


iron 


3.5 


magnesium 


19 


manganese 


0.12 


lead 


0.33 


zinc 


0.14 



Concentration in same river water, 
after being delivered to test 
chambers (mg/1) 

6.8 

2.3 
58 

0.42 

0.05 

0.09 

0.52 

2.2 
20 

0.17 

2.7 

0.62 



It is apparent that the shell deformities and mortalities cannot be 
attributed to the Illinois River water, and that this experiment should be 
repeated after the metal parts in the intake system are replaced with plastic 
fittings. 



Analysis of Shells from Deformed Clams . The deformed shells from clams 
which had been exposed to raw Illinois River water were sent to Dr. Judith 
Murphy, Director of the Center for Electron Microscopy at Southern Illinois 
University for elemental analysis using an X-ray microprobe. As expected, 
the principal component of the shell is calcium in the shell layers which 
were laid down when the clams were still in the Mississippi River. After 
the clams were introduced to the contaminated river water, however, the 



88 



proportion of calcium dropped and silicon became the predominant element. 
The phosphorus and sulfur levels were also somewhat elevated in the deformed 
areas. The microprobe technique does not measure the absolute amount of each 
element present, but the relative amount. At any rate, it is clear that the 
calcium/silicon ratio in the normal part of these shells is almost the re- 
verse of that in the abnormal part. It appears that some contaminant in 
the river water interfered with normal calciiom metabolism during shell 
formation. 



89 



SUMMARY 



1. Two methods for rapidly assessing water quality effects on clams 
were used in this study. One method, which measures particle transport 
rate across the gills of clams, had been used previously in basic research 
on the function of the clam gill. The second method, which measures the 
beating rate of the lateral cilia on the gill, had been used to test the 
effects of drugs on ciliary activity. New methods and equipment for 
measuring the ciliary activity of clams as small as 2 mm in shell length 
were developed as part of this research. 

2. The research also developed culture techniques and chronic and 
acute bioassay methods for use with the fingernail clam, Musculium trans - 
versum , which died out in the Illinois River in the mid- 1 950 's and which 
underwent a population decline in the Keokuk Pool, Mississippi River, in 
1976-77. Following the die-off of fingernail clams, the number of diving 
ducks utilizing the Illinois valley declined from an average of Ih million 
to 160,000, and the abundance and average size of commercially important 
species of fish declined. Results of both the rapid testing methods and 
the chronic and acute bioassays showed that the fingernail clams become 
more sensitive to toxicants and less tolerant of extremes in environmental 
factors as they grow older and larger. This means that it is the older, 
reproductive portion of the population which is most sensitive to environ- 
mental factors. 

3. The ciliary beating rates of gills from two sphaeriacean clams 
( Musculium transversum and Corbicula manilensis ) and the unrelated intertidal 
mussel ( Mytilus edulis ) were noticeably inhibited by light within 1 to 2h hours 



90 



of exposure. Maximum inhibition occurred after 2 to 6 hours exposure. When 
the gill preparations were returned to darkness, the ciliary beating rates 
returned to normal after 2 to 4 hours, with the exception of large Musculium 
transversum , whose ciliary beating rates had not recovered to normal by the 
time the experiment was ended after 4 hours exposure to darkness. Perhaps the 
inhibition of cilia by light is a protective response, should the clam be 
exposed to falling water levels or to the air. The shell of adult fingernail 
clams is translucent, and the shells of small clams are transparent. 

4. Gill preparations from the intertidal mussel, Mytilus edulis , 
maintained their ciliary beating rates over a broader temperature range 

than gill preparations from fingernail clams or Asiatic clams. Small finger- 
nail clams had a broader temperature range than large ones, and the Asiatic 
clam appeared to have the narrowest temperature tolerance, at least as measured 
by the ciliary beating response. Additional experiments would have to be 
performed to determine the acclimation range of these three species of clams, 
and the relationship between the ciliary beating response and acclimation 
temperature. Fingernail clams begin to grow rapidly when water temperatures 
in the Keokuk Pool, Mississippi River, rise above 11-13 C. The ciliary beating 
rate of the fingernail clam substantially increased above 14-15 C, so the 
field results appear to corroborate the laboratory findings. The laboratory 
results also suggest that the ciliary function of fingernail clams would be 
drastically reduced if water temperatures increased above 30 C. 

5. The higher the concentration of dissolved oxygen in the water perfusing 
the gill preparations, the greater the ciliary beating rate. The ciliary 
beating rate of fingernail clams rapidly declined to zero when the dissolved 
oxygen concentration was reduced to 2 ppm. It is unlikely that dissolved 
oxygen concentrations of 2 ppm or lower would immediately kill fingernail clams 



91 



in nature, as the clams can probably switch from aerobic to anaerobic metabolism 
when their shells are closed. The clams have oxygen- sensing organs, the palps, 
located on the gills. When the palps were removed, the gill preparations 
behaved as though they were in water containing little dissolved oxygen, 
even though oxygen levels in the water were maintained at 10 ppm. 

6. The highest concentrations of soditm nitrate and sodium sulfate tested 
had no effect on the ciliary beating rates of gills from large or small clams. 
The maximum nitrate concentration we tested was equivalent to 18 mg/1 as 
nitrogen, and the maximum sulfate concentration tested was 48 mg/1. 

7. Fingernail clam gills were extremely sensitive to copper, lead, 
and zinc, and gills from large clams generally were several orders of 
magnitude more sensitive than gills from small clams. The following con- 
centrations caused 90% reductions in the ciliary beating rates of gills 

from large clams: lead (2 yg/1) , copper (.06-. 6 yg/1) , and zinc (.06-. 65 mg/1). 
Several toxic metals, including lead and copper, occur at higher concentra- 
tions in the Illinois River than in the Mississippi. In view of the 
extreme sensitivity of the clam gills to copper, lead, and zinc, it appears 
that metals in the Illinois River could be a significant stress on adult 
fingernail clams. This tentative conclusion, which is based on tests with 
the sensitive gill preparations, should be verified with additional bioassays 
using intact fingernail clams. 

8. There were significant differences in the responses of gills from 
large (6-11 mm) and small (2-5 mm) clams to: (a) removal and subsequent 
addition of potassium, (b) variation of maintenance dosage of potassium in the 
solution which bathed the gills, and (c) lag period of response to a specific 
dose. Potassium levels required for maintenance of a basal ciliary beating 
rate were 10~ M (39.1 mg/1) for small clams and 10~ M (0.039 mg/1) for 



92 



large clams. Greater concentrations were cilioinhibitory . Lesser concen- 

_o 

trations were generally cilioexcitatory, but concentrations less than 10 M 
(0.00039 mg/1) and 10~^ M (0.000039 mg/1) are insufficient to sustain basal 
rates in large and small clams, respectively. The intact clams were much 
less sensitive to potassium than the gill preparations. The maximum ac- 
ceptable toxicant concentration (MATC) for long-term survival of fingernail 
clams lies between 195 and 275 mg/1 potassium. Potassium concentrations 
below the lethal level actually stimulated the growth of fingernail clams, 
so potassium appears to have no sublethal effects that result in reduced 
growth. However, the effects of potassium on the reproduction of the 
clams was not determined in these experiments. Musculium transversum was 
more sensitive to potassium than fish, about as sensitive as several 
microcrustaceans, and less sensitive than three species of unionid clams. 
The highest potassium concentration in 25 water samples taken near the 
surface of the Illinois River in 1975 was 6 mg/1 potassium, well below the 
lethal threshold. However, potassium concentrations as high as 250 mg/kg 
have been found in the sediments of the Illinois River, so it is possible 
that water associated with these sediments provides considerably higher 
potassium concentrations than water above the sediments. Additional research 
is needed in this area. 

9. Clams exposed to potassium in water with a total hardness equal to 
243 mg/1 as CaCO responded faster than clams tested in water with a total 
hardness equal to 314 mg/1 as CaCO . The toxicity curve of the soft-water 
test approached a vertical asymptote at 400 hours, while the clams tested in 
the hard water did not show a lethal threshold even at 696 hours. 

10. Clams exposed to potassium at a water temperature of 6.5 C died 
more slowly than those in water at a temperature of 16.7 C, but the lethal 



93 



threshold concentration of potassium was not changed. 

11. Large fingernail clams were more sensitive to un-ionized ammonia 
than small fingernail clams, the Asiatic clam ( Corbicula manilensis ) , a 
freshwater unionid mussel ( Elliptio complanata ) , and the intertidal mussel 
( Mytilus edulis ) . An un-ionized ammonia concentration of .03 rag/1 (as 
ammonia nitrogen, NH -N) caused a 50% reduction in the ciliary beating rate 
of gills from large clams, and a concentration of .05-. 06 mg/1 caused com- 
plete inhibition of the cilia. The sensitivity of the gill preparations 

to un-ionized ammonia increased as the oxygen content of the water decreased. 
Results of the chronic bioassay showed that the maximum acceptable toxicant 
concentration (MATC), based on mortality, lies between .34 and .60 mg/1 NH^-N, 
and the MATC based on growth lies between 0.20 and 0.34 mg/1 NH^-N. 

12. Chronic exposure of clams to sublethal concentrations of ammonia 
lowers the tolerance of their gills to a variety of stresses, including addi- 
tional exposure to ammonia. Previous exposure of clams to 0.2 mg/1 NH -N 
for 44 days completely blocked the normal stimulatory response of the gills 
to potassium addition. In fact, the ciliary beating rate was slightly to 
markedly inhibited at all potassium concentrations tested. The gills of the 
clams were sensitized to ammonia by previous exposure to sublethal concentrations 
of ammonia. A 50% reduction in the ciliary beating rate occurred at an un- 
ionized ammonia concentration of .09 mg/1 (NH -N) in control gills not pre- 
viously exposed to ammonia, at .04 mg/1 in gills previously exposed to 0.1 

mg/1 NH„-N for 44 days, and at .01- .02 mg/1 in gills previously exposed to 
0.2 mg/1 NH -N for 44 days. Prior exposure to sublethal concentrations of 
ammonia also reduced both the maximum ciliary response to temperature and the 
temperature tolerance range. Decreasing concentrations of oxygen had a greater 
inhibitory effect on ammonia-exposed gills than on gills not exposed to ammonia. 



94 



13. Concentrations of un-ionized ammonia in the Illinois River in 1975 
were approximately twice the concentrations in the Keokuk Pool, Mississippi 
River. Moreover, the mean and median concentrations at the four Illinois 
River stations where fingernail clams died out were close to the value of 
.03 mg/1 NH -N which caused a 50% reduction in the ciliary beating rate of 
gills from large fingernail clams. 

14. There has been an alarming decrease in the fingernail clam 
populations in the Keokuk Pool, Mississippi River in 1976 and 1977. The 
growth of the surviving clams was also reduced. In 1976, the maximum shell 
length was only 8.3 mm as compared to 12.4 mm in both 1974 and 1975. Finger- 
nail clams begin to reproduce when they are about 5 mm in shell length. 

In 1974 and 1975, the reproductive population numbered 5,000-6,000 clams 
per square meter. In 1976, the reproductive population was reduced to less 
than 1,000 per square meter. 

15. Both the low clam population and reduced growth in Keokuk Pool in 
1976 and 1977 appear to be related to an extremely low river discharge as a 
consequence of a drought in the upper Mississippi River basin. The following 
effects on water quality factors were observed during this low-discharge period: 
lowered dissolved oxygen concentrations, dissolved oxygen stratification, in- 
creased water clarity, and elevation in concentrations of certain materials such 
as un-ionized ammonia. The highest un-ionized ammonia value measured in 
Keokuk Pool in 1976 near Fort Madison, Iowa was 0.198 mg/1 NH -N, which is 

very near the level of 0.20-0.34 mg/1 which affects the growth of fingernail 
clams in our laboratory bioassays after two weeks of continuous exposure. 
The average values of .02-. 03 mg/1 NH„-N in Keokuk Pool were close to, or 
within, the range which caused a 50% reduction in the ciliary beating rate 
of gills from large clams. Our laboratory results also showed that the gills 



95 



become more sensitive to low oxygen levels and ammonia, following chronic 
exposure to un-ionized ammonia concentrations of 0.10 or 0.20 mg/1 NH„-N. 
Since the dissolved oxygen levels on the bottom in Keokuk Pool were reduced 
during the drought, and toxicants, such as heavy metals, were presumably not 
diluted as much in 1976-1977 as in previous years, we hypothesize that 
the combined action of all these factors was sufficient to stress the 
fingernail clams in Keokuk Pool. Our results also showed that as the finger- 
nail clams grow, they become less tolerant of extremes in environmental 
factors such as temperature and low dissolved oxygen, and less tolerant of 
toxicants. We hypothesize that in 1976-1977, the fingernail clams in Keokuk 
Pool grew up to the point where their ciliary activity was impaired by envi- 
ronmental factors. At that point, their ability to feed and respire was 
impaired, and they grew slowly, if at all. Since the clams must reach a size 
of 5 mm to reproduce, and the number of young produced increases as the size 
of the parent increases, the severe reduction in growth also caused a severe 
reduction in reproduction, hence reduced numbers of clams in 1976-1977. This 
interpretation could be confirmed by reproducing the 1976-1977 water quality 
conditions in the laboratory, and observing the effects on clam growth and 
reproduction. 

16. The particle transport rate of gills from large clams was more sensi- 
tive to suspended particles than the transport rate of gills from small clams. 
Sharp silica particles impaired the transport rate of the gills more than 
rounded illite clay particles. The transport rate was reduced at low concentra- 
tions of oxygen. At a constant oxygen concentration of 8 mg/1, a 50% reduction 
in the particle transport rate of gills from small clams was induced by exposure 
to 10 illite clay particles per liter (10.2 mg/1) and 5 x 10 silica particles 



96 



per liter (.002 mg/1) . At the same oxygen concentration, a 50% reduction 

in the particle transport rate of gills from large clams was induced by ex- 

2 3 1 

posure to 10 -10 illite clay particles per liter (.01-. 10 mg/1) and 5 x 10 

silica particles per liter (.002 mg/1). 

17. The beating of cilia on the gills of small fingernail clams was 
almost completely inhibited when the gills were exposed to a sample of water 
taken from the Illinois River on October 5, 1977. Normal ciliary activity 
was maintained when gills were exposed to water from a shallow, sand-point 
well located 100 feet from the Illinois River. Partial inhibition of the 
cilia occurred when the river water was diluted with the well water. 

18. Fingernail clams developed bizarre shell deformities and died 
without reproducing, when they were exposed for four weeks to water con- 
taining a mixture of metals at the following concentrations: 6.8 mg/1 
potassium, 2.3 mg/1 aluminum, 58 mg/1 calcium, 0.42 mg/1 cadmium, 0.05 mg/1 
chromium, 0.09 mg/1 cobalt, 0.52 mg/1 copper, 2.2 mg/1 iron, 20 mg/1 
magnesium, 0.17 mg/1 manganese, 2.7 mg/1 lead, and 0.62 mg/1 zinc. Further 
experiments are needed to determine which of the above metals, or combina- 
tion of metals, produced the shell deformities and mortalities. X-ray micro- 
probe analysis showed that the principal component of the shells was calcium 
in the shell layers which were laid down when the clams were still in the 
Mississippi River. After the clams were introduced to the water containing 

the metals, however, the proportion of calcium in the shell dropped, and silicon 
became the predominant element. Phosphorus and sulfur levels were also somewhat 
elevated in the deformed areas. It appears that some of the above metals inter- 
fere with normal calcium metabolism during shell formation. 



97 



RECOMMENDATIONS 

1. Additional bioassays should be performed to determine whether 
intact fingernail clams are as sensitive to metals as the isolated gill 
preparations. 

2. The effects of raw Illinois River water on intact fingernail 
clams should be determined. If the raw water affects the intact clams, 
additional bioassays should be run in which fingernail clams are exposed 
to river water treated to remove certain components, such as silt, un- 
ionized ammonia, and heavy metals. The results of this deletion bioassay 
would establish whether fingernail clams could survive in the Illinois 
River if certain factors were reduced or removed by waste treatment. 

3. Fingernail clams should be exposed in the laboratory to water 
quality conditions which existed in the Keokuk Pool in 1976-1977, to de- 
termine whether such factors as elevated ammonia levels and lowered dis- 
solved oxygen levels were responsible for the reduced growth and reproduc- 
tion of clams in the Pool in 1976-1977. 

4. The techniques and apparatus for measuring the ciliary beating 
response of gills from clams should be considered as a candidate method 

for rapidly assessing the toxicity of new chemicals, before they are released 
to the aquatic environment. 



98 



RELATION OF THIS RESEARCH TO WATER RESOURCES PROBLEMS 

Fingernail clams play an important role in the ecology of Midwestern 
waters and are sensitive to water quality changes. An unexplained die-off of 
fingernail clams occurred in the Illinois River in 1955, with dramatic ecologi- 
cal repercussions. Similar losses could occur in other waters (there was, in 
fact, a dramatic reduction in fingernail clam populations in the Keokuk Pool, 
Mississippi River, in 1976-1977), unless the reasons for the die-off can be 
determined and prevented. By developing information on the effects of water 
quality on fingernail clams, this research has contributed to the national 
objective of predicting ecologic change and improving water quality. The re- 
search has developed a new method for rapidly assessing water quality effects 
on types of organisms for which no standard method now exists. The rapid 
development of this type of information should be of interest to those who 
must plan and manage water resource systems for a variety of beneficial uses, 
including recreation and the production of fish and waterfowl. The method 
might also be used to test new chemicals before they enter the aquatic environ- 
ment. The technique might also be used to test control agents for pest species, 
such as the introduced Asiatic clam, which enters and clogs condenser tubes of 
power plants. 

Requests for information regarding both the methods and data developed in 
this project have been received from Donovan M. Oseid of the University of Minnesota 
Department of Entomology, Fisheries, and Wildlife, and from Walter Ginsburg, 
Chief Water Bacteriologist of the City of Chicago Water Purification Laboratory. 
There have been 9 requests for reprints of publications resulting from this re- 
search. In addition, project results have been used by the Division of Water 
Resources, Illinois Department of Transportation, the Illinois Department of Con- 
servation, and the U.S. Fish and Wildlife Service in reviewing permits for 
development along the Keokuk Pool, Mississippi River. 



99 



ACKNOWLEDGEMENTS 

We are grateful to the Division of Fisheries, Illinois Department of Con- 
servation, and to Mr. Hubert Bell, Supervisor of the Fisheries Building at 
Havana, for providing laboratory space and assisting with the construction of a 
pipeline to bring Illinois River water into the laboratory. The Supporting 
Services of the Illinois Natural History Survey, including Robert 0. Ellis, 
Assistant for Operations, and Larry D. Gross and Jerry McNear, Operations 
Assistants, also helped install the pipeline at the laboratory. Dr. Kenneth E. 
Smith and Dr. Allison Brigham, Illinois Natural History Survey at Urbana, analyzed 
water samples from the Mississippi and Illinois Rivers and from the laboratory 
bioassays. Dr. Dean Wesley, Department of Agriculture at Western Illinois Uni- 
versity, provided a flame photometer and instruction for some chemical analyses. 
Mr. Tony Petrovich, owner and manager of Tony's Plumbing and Heating, Inc., in 
Alpha, Illinois, provided instruction and parts for construction of diluters and 
plumbing in the bioassay laboratories at Havana. Mr. Carl M. Thompson and Mr. 
Michael J. Sandusky provided much-needed assistance in both the laboratory and 
field. Mr. Harold Henderson helped count and measure fingernail clams during the 
chronic bioassays, and typed and edited the final report. Dr. Judith Murphy, Di- 
rector of the Center for Electron Microscopy at Southern Illinois University, Car- 
bondale, provided an elemental analysis of deformed clam shells, using electron 

microprobe techniques. We are grateful to Mr. A. A. Fitzsimmons and Mr. John L. 

Maier of Chevron Chemical Company, Fort Madison, Iowa, for supplying us with water 

quality data from their intake on Keokuk Pool. Finally, we are grateful to Dr. 

Glenn Stout, Director of the Water Resources Center at the University of Illinois, 

for helpful advice in the preparation of the initial research proposal, and for 

encouragement and understanding during the project and during preparation of the 

final report. 

The work upon which this publication is based was supported by funds 
provided by the U.S. Department of the Interior as authorized under the 
Water Resources Research Act of 1965, P.L. 88-379. 



100 



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Gale, W.F. 1973. Predation and parasitism as factors affecting Sphaerium 

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104 
APPENDIX A. TEST CONDITIONS AND RESULTS OF ACUTE AND CHRONIC BIOASSAYS. 



Table 7. Test Conditions for Adult Test Al . 











Dissolved 


Total 


' Pot 
(m 

Mean 




Test 
Chamber 


Temperature 
(C) 
Mean Range 


PH» 


Oxygen 

(mg/l) 

Mean Range 


Alkalinity 

(mg/l) 

as CaCO^) 


assium 
g/1) 

Range 


1 


16.7 


16.0-17.0 


8.50 


9.3 


8.8-9.7 


161 


581.0 


562.0-600.0 


2 


16.7 


16.0-17.0 


8.50 


9.3 


8.5-9.7 


161 


316.5 


288.0-345.0 


3 


16.7 


16.0-17.0 


8.50 


9.3 


8.6-9.7 


161 


192.0 


181.0-203.0 


4 


16.7 


16.0-17.0 


8.50 


9.3 


8.6-9.6 


161 


106.3 


102.5-110.0 


5 


16.7 


16.0-17.0 


8.50 


9.3 


8.8-9.7 


161 


57.3 


48.0- 66.5 


6 


16.7 


16.0-17.0 


8.50 


9.3 


8.8-9.7 


161 


37.0 


34.0- 40.0 


7 16.7 
(control) 


16.0-17.0 


8.50 


9.3 


8.9-9.8 


161 


9.5 


7.3- 11.7 



^Measured only once during the bioassay. 



Table 8 . Results of Test Al. 



Mean Potassium 
Concentration 
(mg/l) 


Hr 
48 


Percent Dead 
Hr 
72 


at 

Hr 
96 


Hr 
120 


581.0 


55.6 


85.7 


100.0 


100.0 


316.5 


27.2 


60.0 


80.0 


90.0 


192.0 


12.5 


37.5 


50.0 


50.0 


106.3 


11.1 


11.1 


22.2 


22.2 


57.3 





10.0 


10.0 


10.0 


37.0 





10.0 


10.0 


10.0 


9.5 
(control) 















LC50 



518 



255 



185 



168 



95 percent 
confidence limits 



310- 
865 



175- 
372 



128- 
268 



119- 
237 



slope function 



2.66 



2.42 



2.06 



1.94 



105 



Table 9. Test Conditions for Adult Test A2. 



Test 
Chamber 


Temperature 

(C) 
Mean Range 


PH 
Mean Range 


Dissolved 

Oxygen 

(mg/l) 

Mean Range 


Total 
Alkalinity 

(mg/l 

as CaC03) 

Mean Range 


Potassium 

(mg/l) 

Mean Range 




17.0 


16.3-17.3 


8.22 


8.18-8.28 


9.3 


9.1-9.4 


154 


152-156 


2036.0 


1956.0-2116.0 




17.0 


16.1-17.3 


8.23 


8.20-8.28 


9.3 


9.2-9.4 


155 


154-156 


1139.0 


1102.0-1176.0 




16.8 


15.9-17.1 


8.27 


8.25-8.30 


9.3 


9.2-9.4 


158 


156-160 


606.0 


599.0- 613.0 




16.7 


15.7-17.0 


8.24 


8.18-8.28 


9.2 


9.0-9.4 


154 


152-156 


356.0 


349.0- 360.0 




16.9 


15.9-17.3 


8.25 


8.18-8.32 


9.3 


9.1-9.4 


154 


150-160 


190.3 


177.8- 211.2 




17.0 


16.0-17.4 


8.26 


8.12-8.41 


9.3 


9.2-9.4 


155 


153-156 


108.1 


106.0- 109.6 




16.9 


15.9-17.3 


8.22 


8.15-8.38 


9.2 


9.0-9.4 


155 


152-156 


64.1 


63.3- 64.7 


8 17.0 
(control) 


16.0-17.5 


8.23 


8.13-8.28 


9.2 


9.0-9.4 


152 


152-153 


2.6 


2.0- 3.0 



Table 10. Results of Test A2. 



Mean Potassium 

Concentration 

(mg/l) 


Hr 

48 




Hr 
72 




Hr 
96 


Percent Dead 
Hr 
144 


at 


Hr 
192 




Hr 
240 




Hr 
312 




2036.0 


95 (94. 


7)' 


'lOO 




100 




100 




100 




100 




100 




1139.0 


75 (73. 


7) 


100 




100 




100 




100 




100 




100 




606.0 


25 (21. 


0) 


100 




100 




100 




100 




100 




100 




356.0 


5 (0) 




45 (42. 


1) 


80 


(78.9) 


95 (94.7) 


100 




100 




100 




190.3 


(0) 




10 ( 5. 


3) 


15 


(10.5) 


25 (21. 


1) 


40 (36.8) 


50 (47, 


.4) 


50 (47. 


.4) 


108.1 


5 (0) 




5 (0) 




5 


(0) 


15 (10. 


5) 


15 (10. 


5) 


15 (10.5) 


15 (10. 


.5) 


64.1 


5 (0) 




10 ( 5.3) 


10 


( 5.3) 


10 ( 5. 


3) 


10 ( 5. 


3) 


10 ( 5. 


.3) 


10 ( 5. 


3) 


2.6 

(control) 


5 (0) 




5 (0) 




5 


(0) 


5 (0) 




5 (0) 




5 (0) 




5 (0) 




LC50 


880 




370 




280 




228 




212 




200 




200 




95 percent 
confidence 
limits 


707- 
1094 




293- 
467 




229- 
342 




185- 
281 




167- 
270 




156- 
257 




156- 
257 




slope function 


1.65 




1.46 




1.38 




1.40 




1.48 




1.78 




1.78 





iNinnbers in parentheses indicate values corrected for control mortality. 



106 



Table 11. Test Conditions for Juvenile Test Jl. 



Temperature 
Test (C) 



Dissolved Total 

Oxygen Alkalinity Potassium 

(mg/1) (mg/1 (mg/1) 



5 

6 

7 
(control) 



17,0 16.9-17.2 8.47 8.40-8.50 9.2 9.0-9.3 

17.0 16.9-17.2 8.49 8.A0-8.55 9.2 9.1-9.2 

17.0 16.9-17.2 8.A9 8.45-8.55 9.2 9.1-9.3 

17.0 16.9-17.2 8.50 8.45-8.55 9.2 9.0-9.3 

17.0 16.9-17.2 8.51 8.50-8.55 9.2 9.0-9.4 

17.0 16.9-17.2 8.49 8.45-8.50 9.2 9.0-9.3 

17.0 16.9-17.2 8.49 8.45-8.50 9.2 9.1-9.3 



161 
161 
161 



3532.0 3300-3680 

2103.0 1920-2220 

1221.0 1080-1310 

536.8 505- 555 

393.6 360- 415 

224.1 200- 240 

8.1 7.3- 8.6 



pleasured once during the bloassay. 



Table 12. Results of Test Jl. 



Mean Potassium 
Concentration 
(mg/1) 


Hr 
48 


Hr 
72 


Hr 
96 


Hr 
120 


Percent I 

Hr 
168 


lead at 

Hr 
240 


Hr 
264 


Hr 
293 


Hr 
312 


3532.0 


66.7 


88.9 


100 


100 


100 


100 




100 


100 


100 


2103.0 


10.0 


40.0 


100 


100 


100 


100 




100 


100 


100 


1221.0 


10.0 


30.0 


100 


100 


100 


100 




100 


100 


100 


536.8 


10.0 


20.0 


30 


60 


90 (88)^ 


100 




100 


100 


100 


393.6 





10.0 


30 


30 


80 (75) 


80 


(75) 


90 (87.5) 


90 (87.5) 


90 (87. i 


224.1 


20.0 


20.0 


30 


30 


40 (25) 


40 


(25) 


60 (50) 


60 (50) 


60 (50) 


7.3 
(control) 














20 (0) 


20 


(0) 


20 (0) 


20 (0) 


20 (0) 


LC50 


2700 


1680 


520 


435 


300 


270 




250 


250 


250 


95 percent 
confidence 
limits 


1753- 
4158 


960- 
2940 


366- 
738 


292- 
648 


225- 
399 


213- 
343 




189- 
330 


189- 
330 


189- 
330 


slope function 


1.63 


1.88 


1.49 


2.23 


1.59 


1.47 




1.37 


1.37 


1.37 



^Numbers In parentheses indicate values corrected for control mortality. 



107 
Table 13. Test Conditions for Juvenile Test J2. 



Total 

Dissolved Alkalinity 

Temperature „ Oxygen (mg/l Potasaltun 

Test (C) ^*^ (mg/l) as CaCOa) (mg/l) 

Chamber Mean Range Mean Range Mean Range Mean Range Mean Range 

1 16.6 15.8-17.0 8.11 8.10-8.12 9.3 9.1-9.5 140 128-152 3244.0 3236.0-3252.0 

2 16.7 15,7-16.8 8.21 8.13-8.28 9.3 9.1-9.5 155 152-158 2062.0 2000.0-2124.0 

3 16.5 15.6-16.9 8.26 8.18-8.35 9.3 9.1-9.6 157 156-160 1101.0 1100.0-1102.0 

4 16.6 15.7-16.9 8.26 8.18-8.35 9.4 9.0-9.6 157 156-161 613.0 599.0- 621.0 

5 16.8 15.9-17.0 8.26 8.20-8.37 9.3 9.0-9.5 154 152-156 361.3 352.0- 371.0 

6 16.8 16.0-17.1 8.28 8.18-8.35 9.3 9.0-9.5 156 154-158 196.3 175.8- 209.2 

7 16.8 16.0-17.1 8.31 8.25-8.35 9.2 9.0-9.4 159 156-160 2.8 2.1- 3.5 
(control) 



Table 14. Results of Test J2. 



Mean Potassium 

Concentration 

(mg/l) 


Hr 
48 


Hr 
72 


Hr 
96 




Percent Dead at 

Hr Hr 
144 192 


Hr 
240 


Hr 
312 


Hr 
384 


3244.0 


55 


95 


100 




100 


100 


100 


100 


100 


2062.0 


5 


45 


90 (89. 


,5)^ 


100 


100 


100 


100 


100 


1101.0 


10 


10 


45 (42. 


.1) 


95 (94.7) 


100 


100 


100 


100 


613.0 


5 


10 


25 (21. 


.1) 


75 (73.7) 


95 (94.4) 


100 


100 


100 


361.3 


15 


20 


25 (21. 


.1) 


30 (26.3) 


60 (55.6) 


75 (70.6) 


85 (82.4) 


90 (87.5) 


196.3 








(0) 




(0) 


10 (0) 


10 (0) 


10 (0) 


10 (0) 


2.8 
(control) 








5 (0) 




5 (0) 


10 (0) 


15 (0) 


15 (0) 


20 (0) 


LC50 


3150 ^ 


1960 


1000 




510 


350 


320 


310 


300 


95 percent 
confidence 
limits 




1574- 
2440 


775- 
1290 




419- 
621 


280- 
437 


283- 
362 


271- 
355 


261- 
345 


slope function 




1.42 


2.04 




1.57 


1.43 


1.22 


1.24 


1.26 



Numbers In parentheses Indicate values corrected for control mortality. 
'Chl-square could not be determined. 



108 



Table 15. Test Conditions for Juvenile Test J3. 



Test 
Chamber 


Temperature 
(C) 
Mean Range 


Mean 


PH 

Range 


Dissolved 

Oxygen 

(mg/l) 

Mean Range 


Total 

Alkalinity 

(ag/1 

as CaCOj) 

Mean Range 


Potassium 
(mg/1) 
Mean Range 




16.8 


16.6-17.3 


8.64 


8.60-8.68 




8.9- 9.6 


23A 


228-238 


3166.7 


3072.0-3248.0 




16.9 


16.6-17.3 


8.65 


8.61-8.67 




8.8- 9.7 


242 


232-251 


1909.3 


1832.0-1980,0 




16.9 


16.6-17.3 


8.66 


8.61-8.69 




8.8- 9.8 


239 


216-255 


1022.3 


960.0-1076.0 




16.9 


16.6-17.2 


8.65 


8.60-8.69 




8.8- 9.9 


250 


204-255 


557.8 


515.0- 600.0 




16.8 


16.6-17.1 


8.65 


8.60-8.68 




8.7-10.0 


219 


188-251 


332.0 


308.0- 352.0 




16.8 


16.5-17.1 


8.65 


8.60-8.68 




9.1- 9.8 


221 


196-248 


181.3 


160.8- 196.0 


7 16.7 
(control) 


16.A-17.0 


8.66 


8.62-8.70 




8.8-10.2 


240 


216-259 


4.7 


2.4- 10.0 



Table 16. Results of Test J3. 



Mean Potassium 










Percent Dead 


at 












Concentration 


Hr 


Hr 


Hr 


Hr 


Hr 


Hr 


Hr 


Hr 


Hr 


Hr 


Hr 


Hr 


(mg/1) 


96 


120 


144 


168 


192 


240 


312 


384 


456 


528 


600 


696 


3166.7 


(64.7)^ 


90 
(88.2) 


100 


100 


100 


100 


100 


100 


100 


100 


100 


100 


1909.3 


40 
(29.4) 


60 
(52.9) 


100 


100 


100 


100 


100 


100 


100 


100 


100 


100 


1022.3 


25 


30 


50 


65 


70 


80 


95 


100 


100 


100 


100 


100 




(11.8) 


(17.6) 


(A1.2) 


(58.8) 


(64.7) 


(75) 


(93.8) 













557.8 
332.0 
181.3 



4.7 
(control) 



15 



15 



15 



15 



15 



30 



55 



95 



(0) (0) (0) (0) (0) (0) (12.5) (38.1) (58.8) (93.1) (93.1) (91.9) 

10 15 15 20 20 25 25 30 45 55 65 75 
( ) ( ) ( ) ( 5.9) ( 5.9) ( 6.3) ( 6.3) ( 3.8) (24.0) (38.1) (51.9) (59.5) 



10 20 20 20 20 20 22.2 
( ) ( 5.9) ( 5.9) ( 5.9) ( 5.9) ( ) ( 2.8) 



22.2 22.2 22.2 33.3 33.3 
( ) ( ) ( ) ( 8.3) ( ) 



15 15 15 15 15 20 
(0) (0) (0) (0) (0) (0) 



20 27.25 27.25 27.25 27.25 38.35 
(0) (0) (0) (0) (0) (0) 



95 percent 
'Confidence 

limits 

slope function l.{ 



1678- 


1118- 


931- 


845- 


784- 


708- 


543- 


475- 


403- 


364- 


258- 


260- 


3725 


2898 


1231 


1137 


1092 


1020 


863 


661 


571 


407 


397 


394 



Numbers in parentheses indicate values corrected for control mortality. 



109 



Table 17. Test Conditions for Juvenile Test J4. 



Total 
Dissolved Alkalinity 

Temperature „ Oxygen (mg/l Potassium 

Test (C) P" (mg/l) as CaCOs) (mg/l) 

Chamber Mean Range Mean Range Mean Range Mean Range Mean Range 



6. A 6.0-6.9 8.37 8.35-8.38 11.6 11.2-12.2 207 206-208 321A.0 3196.0-3232.0 

6.5 6.2-7.0 8.41 8.40-8.41 11.6 11.0-12.2 213 208-220 1894.0 1876.0-1912.0 

6.6 6.0-7.0 8.42 8.41-8.42 11.5 11.0-11.8 212 210-214 1052.0 1046.0-1058.0 

6.7 6.1-7.0 8.44 8.42-8.45 11.4 10.8-11.6 212 210-212 571.5 562.0- 581.0 
6.7 5.2-6.8 8.46 8.40-8.55 11.5 11.0-11.8 204 200-208 348.7 341.0- 355.0 
6.5 5.2-7.1 8.41 8.32-8.49 11.2 11.0-12.0 218 212-220 9.2 2.4- 12.9 



(control) 



Table 18. Results of Test J4. 















Dead at 










Mean Potassium 






















Concentration 


Hr 


Hr 


Hr 


Hr 


Hr 


Hr 


Hr 


Hr 


Hr 


Hr 


(mg/l) 


72 


96 


144 


192 


240 


312 


384 


456 


552 


648 


3214 


60 1 
(57.9) 


85 


95 


100 


100 


100 


100 


100 


100 


100 




(84.2) 


(94.4) 
















1894 


40 
(36.8) 


55 
(52.6) 


95 
(94.4) 


100 


100 


100 


100 


100 


100 


100 


1052 


10 
( 5.3) 


30 
(26.3) 


50 
(44.4) 


60 
(55.6) 


85 
(83.3) 


100 


100 


100 
2 


100 
2 


100 

2 



571.5 
348.7 



9.2 
(control) 



95 percent 
confidence 

limits 



10 25 45 70 
(0) (0) (0) (0) (16.7) (35.3) (64.7) 



25 



30 



35 



35 



40 



45 



50 



( 5.3) ( 5.3) (16.7) (16.7) (22.2) (23.5) (23.5) (29.4) (35.3) (41.2) 

5 5 10 10 10 15 15 15 15 15 
(0) (0) (0) (0) (0) (0) (0) (0) (0) (0) 



1180 1020 



2046- 1313- 1009- 
3564 2537 1381 



894- 709- 
1164 996 



406- 
713 



410- 
497 



344- 
563 



310- 
516 



slope function 1.89 1.70 1.29 1.24 1.32 



388 

301- 
500 

1.52 



lumbers in parentheses Indicate values corrected for control mortality. 
Test chamber contaminated. 



110 



Table 19. Test Conditions for Chronic Bioassay Kl , 



Test 
Chamber 


Temperature 
(C) 


Dissolved 
mg/1 


1 Oxygen 
Z Saturation 


pH 


Total 
Alkalinity 
mg/1 CaCOj 


Potassium 
mg/1 K 


6 


22.9 + 0.81^ 


7.19 + 0.391 


84.7 + 3.97 


8.03 + 0.063 


169 + 2.3 


195 + 12.73 




(21,4-24.4) 


(6.30-8.30) 


(75.7-97.4) 


(7.90-8.14) 


(164-172) 


(186-205) 


5 


22.9 + 0.78 


7.36 + 0.294 


86.7 + 3.26 


8.02 + 0,063 


170 + 3.3 


115^ 




(21.6-24.3) 


(6.65-7.90) 


(77.7-93.7) 


(7,89-8,11) 


(164-176) 




4 


23.0 + 0.80 


7.28 + 0.290 


85.8 + 2.61 


8.02 + 0.054 


170 + 2.4 


69.5+ 2.12 




(21.6-24.4) 


(6.64-7.85) 


(79.8-91.3) 


(7,90-8,09) 


(167-176) 


(68.0-71.0) 


3 


23.0 + 0.82 


7.38 + 0.310 


87.1 + 2.92 


8,03 + 0,057 


169+1.7 


43,6 + 0.07 




(21.6-24.4) 


(6.52-7.90) 


(78.4-91,5) 


(7,90-8,13) 


(168-172) 


(43,5-43.6) 


2 


23.0 + 0.85 


7,29 + 0.306 


86,0 + 2,74 


8,03 + 0,056 


170 + 2.4 


30.5^ 




(21.4-24.4) 


(6.55-7.95) 


(78.7-91.2) 


(7.90-8.12) 


(166-175) 




1 


23.0 + 0.86 


6.95 + 0.540 


82,0 + 5,57 


7.97 + 0.068 


169 + 2.2 


11.9 + 1.59 


(control) 


(21.4-24.4) 


(6.08-8.04) 


(72.4-92.2) 


(7.89-8.15) 


(168-175) 


(10.8-13.0) 



Mean + standard deviation. 
2 (Range) 
Only one sample analyzed. 



Table 20. Results of Chronic Bioassay Kl 



Potassium 
Test Concentration 
Chamber mg/1 



)ays of Exposure 

14 28 



195 + 12,7 
(186-204) 



69.5 + 2.12 
(68,0-71,0) 



43.6 + 0.07 
(43.5-43.6) 



11,9 + 1,59 
(10.8-13,0) 



Mean Length mm 
Total Mortality X 
Mean Length mm 
Total Mortality X 
Mean Length mm 
Total Mortality X 
Mean Length mm 
Total Mortality X 
Mean Length mm 
Total Mortality X 
Mean Length mm 
Total Mortality X 



2.6+0.36^ 3.4+0.55 3.6+0.59 3.7+0.60 

(1.9-3.2) (1.9-4.8) (1.9-5,0) (1.9-5.1) 

7.5 15 41.3 

2.6+0.34 3.5+0.56 3.8+0.58 4.1+0.57 

(1.8-3.1) (2.3-4.9) (2.8-5.3) (3.1-5.2) 

10,4 15.6 48,8 

2.5+0,35 3,4+0.51 3.5+0.52 3.7+0.63 

(2.0-3.5) (2,5-4,9) (2.6-4.9) (2.7-5.1) 

11,2 14.0 30.0 

2.6 + 0.303 
(1.8-3.0) 



2.5+0,33 3,5+0,51 3,6+0,48 3,7+0.55 

(1.9-3.2) (2.6-4.3) (2,7-4.6) (2.7-4.5) 

5.0 30.0 

2.6+0.28 3.4+0,40 3.5+0.39 3.7+0,36 

(2.1-3.1) (2,6-4,0) (2,6-4,1) (3,0-4,2) 

10,5 18,5 67,4 



iMean + standard deviation (Range) . 

^Only one sample analyzed, 

^tank contaminated, eliminated from test. 



Ill 



Table 21. Test Conditions for Chronic Bioassay K2. 



Test 
Chamber 


Temperature 
(C) 


Dlssolveci 
mg/1 


1 Oxygen 
X Saturation 


pH 


Total 
Alkalinity 
mg/l CaCOg 


Potassium 
mg/l K 




23.9 ♦ 0.71 
(21.8-24.7) 


7.60 + 0.496 
(6.49-8.70) 


91.3 + 5.78 
(76.1-100.6) 


8.14 + 0.083 
(7.92-8.29) 


167 + 3.6 
(162-172) 


2752 




24.0 + 0.74 
(21.7-24.7) 


7.69 + 0.493 
(5.99-8.64) 


92.4 + 5.95 
(70.1-101,7) 


8.14 + 0.083 
(7.91-8.28) 


170 + 4.4 
(164-176) 


184 




24.0 + 0.73 
(21.7-24.7) 


7.67 + 0.470 
(6.20-8.62) 


92.1 + 5.61 
(72.6-100.4) 


8.13 + 0.085 
(7.90-8.29) 


168 + 4.5 
(162-174) 


106 




23.9 + 0.72 
(21.7-24.7) 


7.71 + 0.504 
(6.07-8.80) 


92.7 + 6.07 
(71.1-101.6) 


8.14 + 0.094 
(7.88-8.29) 


167 + 3.7 
(163-173) 


65 




23.9 + 0.72 
(21.7-24.7) 


7,68 + 0.486 
(6.20-8.74) 


92.1 + 5.76 
(72.7-101.6) 


8,14 + 0.088 
(7.89-8.29) 


168 + 5.1 
(162-176) 


45 


1 
(control) 


23.8 + 0.67 
(21.8-24.5) 


7.74 + 0.465 
(6.55-8.70) 


92.7 + 5.40 
(76.8-100.6) 


8.12 + 0.094 
(7.89-8.29) 


167 + 4.4 
(162-172) 


14.3 



Mean + standard deviation (Range) 
i>nly one sample analyzed. 



Table 22. Results of Chronic Bioassay K2. 



Potassium 
Test Concentration 

Chamber mg/l 



Days of Exposure 
14 



28 



(control) 



Mean Length mm 
Total Mortality % 
Mean Length mm 
Total Mortality X 
Mean Length mm 
Total Mortality X 
Mean Length 
Total Mortality X 
Mean Length mm 
Total Mortality X 

Mean Length 
Total Mortality Z 



2.5 + 0.27 
(1.9-3.2) 



2.6 + 0.25 
(2.1-3.2) 



2.5 + 0.23 
(2.1-3.1) 



2.6 + 0.27 
(2.1-3.2) 



2.5 + 0.23 
(2.2-3.0) 



2.6 + 0.26 
(2.2-3.2) 



2.6 + 0.28 2.7 + 0.45 

(2.2-3.3) (2.0-3.3) 

40 84.2 

3.1 + 0.40 3.4 + 0.48 

(2.3-3.9) (2.3-4.5) 

5.5 13.2 

3.2+0.41 3.9+0.59 

(2.2-4.1) (2.3-4.9) 

2.5 12.8 

3.3 + 0.47 3.4 + 0.48 

(2.3-4.5) (2.4-4.5) 

2.6 10.3 

3.0 + 0.33 3.3 + 0.40 

(2.3-3.7) (2.6-4.2) 

5.0 37.5 

3.0+0.29 3.1+0.25 

(2.2-3.5) (2.6-3.4) 



lOnly one sample analyzed. 

4lean + standard deviation (Range) . 



112 



Table 23. Test Conditions for Chronic Bioassay NH 2 . 

Total Total Undlssoclated 

Test Temperature Dissolved Oxygen Alkalinity Free CO Ammonia Ammonia 

Chamber (C) mg/1 X Saturation pH mg/1 CaCO^ mg/1 ^ mg/1 NH^-N mg/1 NH^-N 

g 23.5+0.45 6.75+0.728 80.3+8.50 8.09+0.161 165+3.0 3.12+0.420 16.16+2.219 0.93+0.304 

(22.9-24.4) (5.02-7.95) (59.6-94.0) (7.88-8.55) (160-168) (2.46-3.73) (12.84-19.88) (0.46-1.53) 

J 23.5+0.45 7.20+0.643 85.6+7.40 8.15+0.116 168+3.1 2.40+0.280 8.83+1.514 0.59+0.144 

(22.8-24.4) (5.91-8.20) (70.0-96.5) (7.92-8.49) (163-172) (2.12+2.94) (5.80-Ll. 27) (0.34-0.82) 

^ 23.5+0.47 7.38+0.567 87.8+6.32 8.16+0,099 168+2.1 2.40+0.410 5.07+0.909 0.35+0.089 

(22.9-24.4) (6.00-8.19) (72.6-96.2) (7.94-8.37) (163-171) (1.98-3.31) (2.89-6.20) (0.17-0.49) 

3 23.4+0.46 7.44+0.527 88.5+5.80 8.14+0.071 168+1.6 2.42+0.250 3.03+0.575 0.20+0.051 

(22.2-24.4) (6.24-8.20) (75.4-96.4) (7.96-8.35) (166-171) (1.96-2.80) (1.80-3.78) (0.11-0.27) 

2 23.4+0.47 7.58+0.424 90.1+4.68 8.16+0.055 168+1.9 2.29+0.194 1.48+0.326 0.10+0.030 

(22.8-24.4) (6.90-8.28) (82.3-97.2) (8.04-8,30) (167-172) (1.96-2.60) (0.82-1.92) (0.05-0.13) 



(con- 
trol) 



23.5+0.47 7.84+0.315 92.9+3.02 8.20+0.041 169+2.2 1.99+0.185 0.10+0.026 0.01+0.002 
(22.8-24.5) (7.45-8.52) (87.3-97.4) (8.13-8.33) (168-172) (1.54-2.15) (0.06-0.14) (0.004-0.01) 



Mean + standard deviation (Range) . 





Table 2A , Results of Chron 


ic Bioassay 


NH22. 


Test 
Chamber 




Days 



f Exposure 
28 


42 




Interval Undlssoclated 
Ammonia mg/1 NHj-N 


- 


1.01 + 0.300 
(0.64-1.53) 


• 0.93 + 0.304 
(0.46-1.53) 


6 


Mean Length mm 


2.3 + 0.28^ 
(1.7-2.9) 


2.3 + 0.25 
(1.9-2.7) 


2.3 + 0.31 
(2.0-2.7) 




Total Mortality % 


- 


49.5 


92.2 




Interval Undlssoclated 
Ammonia ng/1 NH^-N 


- 


0.62 + 0.143 
(0.45-0.82) 


0.59 + 0.144 
(0.34-0.82) 


5 


Mean Length mm 


2.3 + 0.28 
(1.8-2.8) 


2.3 + 0.27 
(1.9-2.9) 


2.2 + 0.29 
(1.8-2.9) 




Total Mortality Z 


- 


28.7 


66.6 




Interval Undlssoclated 
Ammonia mg/1 NH3-N 


- 


0.34 + 0.085 
(0.17-0.43) 


0.35 + 0.089 
(0.17-0.49) 


4 


Mean Length mm 


2.2 + 0.19 
(1.8-2.7) 


2.3 + 0.20 
(1.8-2.7) 


2.3 + 0.20 
(2.0-2.7) 




Total Mortality X 


- 


7.9 


21.6 














Interval Undlssoclated 
Ammonia mg/1 NH^-N 


- 


0.21 + 0.510 
(0.11-0.27) 


0.20 + 0.051 
(0.11-0.27) 


3 


Mean Length mm 


2.2 + 0.24 
(1.8-2.7) 


2.4 + 0.23 
(1.9-2.8) 


2.4 + 0.26 
(1.9-2.9) 




Total Mortality % 


- 


6.3 


10.0 




Interval Undlssociated 
Ammonia mg/1 NH^-N 


- 


0.10 + 0.031 
(0.05-0.13) 


0.10 + 0.030 
(0.05-0.13) 


2 


Mean Length mm 


2.1 + 0.20 
(1.8-2.6) 


2.4 + 0.27 
(1.8-3.0) 


2.6 + 0.32 
(1.8-3.3) 




Total Mortality X 


- 


7.5 


13.2 




Interval Undlssoclated 
Ammonia mg/1 NH^-N 


- 


0.01 + 0.002 
(0.005-0.01) 


0.01 + 0.002 
(0.004-0.01) 


(control) 


Mean Length mm 


2.2 + 0.32 
(1,8-2.8) 


2.3 + 0.33 
(1.8-3.1) 


2.3 + 0.32 
(1.8-3.1) 




Total Mortality X 


- 


15.0 


21.6 



lean + standard deviation (Range). 



113 



Table 25. Test Conditions for Chronic Bioassay NH„3. 



Test Temperature 
Chamber (C) 



Dissolved Oxygen 
mg/1 Z Saturation 



Total 
Alkalinity 
mg/1 CaCO 



Total 
Ammonia 
mg/1 MB,-N 



Undlsaoclated 
Ammonia 
mg/1 NH,-N 





23.0 + 0.2A 
(22.2-23.4) 


6.55 + 0.768 
(5.35-8.40) 


77.1 + 9.14 
(63.5-99.3) 


8.14 + 0.072 
(7.98-8.30) 


166 ± 4.1 
(158-171) 




22.9 + 0.28 
(22.0-23.4) 


6.82 + 0.716 
(5.70-8.48) 


80.2 + 8.61 
(66.9-100.2) 


8.12± 0.061 
(7.99-8.28) 


166 + 5.0 
(158-173) 




22.9 + 0.26 
(22.2-23.4) 


7.12 + 0.658 
(5.80-8.50) 


83.9 ± 7.89 
(68.1-100.8) 


8.10 + 0.065 
(7.98-8.29) 


156 ± 5.4 
(158-172) 




22.9 + 0.24 
(22.2-23.3) 


7.14 + 0.649 
(6.20-8.55) 


84.1 + 7.79 
(72.7-101.3) 


8.08 + 0.066 
(7.97-8.28) 


164 + 5.5 
(159-172) 




22.9 + 0.27 
(22.1-23.3) 


7.34 + 0.614 
(6.30-8.59) 


86.5 + 7.35 
(75.2-101.8) 


8.09 ± 0.068 
(7.98-8.28) 


165 + 5.8 
(158-173) 


(con- 
trol) 


23.0 ± 0.32 
(22.0-23.3) 


8.11 ± 0.365 
(7.50-8.80) 


95.6 ± 4.53 
(88.2-104.3) 


8.18 ± 0.060 
(8.03-8.30) 


165 ± 3.8 
(161-171) 



2.27 + 0.387 
(1.96-3.07) 

2.32 + 0.310 
(1.82-2.76) 

2.35 + 0.350 
(1.77-2.81) 

2.43 + 0.410 
(1.83-2.89) 

2.40 + 0.420 
(1.79-2.85) 

2.06 + 0.320 
(1.74-2.71) 



18.04 ± 0.650 
(16.1-18.91) 



9.51 + 0.516 
(8.33-10.17) 



5.51 ± 0.288 
(4.79-5.87) 



3.33 + 0.211 
(2.88-3.61) 



1.59 + 0.159 
(1.29-1.80) 



0.08 ± 0.022 
(0.04-0.13) 



1.20 ± 0.197 
(0.84-1.53) 

0.60 ± 0.097 
(0.43-0.74) 

0.34 + 0.062 
(0.27-0.46) 

0.20 + 0.040 
(0.14-0.27)1 

0.10 + 0.023 
(0.07-0.14) 

0.01 ± 0.002 
(0.003-0.01) 



Wm + 



standard deviation (Range). 



Table 26, 



Results of Chronic Bioassay NH„3. 



Test 
Chamber 







Days of 
14 


Exposure 
28 


42 




Interval Undlssoclated 
Ammonia mg/1 NH3-K 




1.17 + 0.202 
(0.84-1.32) 


1.21 + 0.189 
(0.84-1.53) 


1.20 + 0.197 
(0.84-1.53) 


6 


Mean Length mm 


2.5 + 0.20^ 
(2.1-2.9) 


2.5 + 0.19 
(2.2-2.7) 


2.5 + 0.15 
(2.3-2.7) 


- 




Total Mortality X 


- 


33.9 


86.7 


100 




Interval Undlssoclated 
Ammonia mg/1 NH3-N 


- 


0.63 + 0.126 
(0.48-0.74) 


0.62 + 0.097 
(0.48-0.74) 


0.60 + 0.097 
(0.43-0.74) 


5 


Mean Length mn 


2.5 + 0.26 
(2.1-3.1) 


2.5 + 0.22 
(2.1-3.1) 


2.5 + 0.22 
(2.1-3.1) 


2.5 + 0.12 
(2.2-2.6) 




Total Mortality I 


- 


10 


25 


71.7 




Interval Undlssoclated 
Ammonia mg/1 NH3-N 


- 


0.38 + 0.076 
(0.28-0.46) 


0.35 + 0.066 
(0.28-0.46) 


0.34 + 0.062 
(0.27-0.46) 


4 


Mean Length mm 


2.4 + 0.19 
(2.1-2.8) 


2.5 + 0.19 
(2.1-2.9) 


2.5 + 0.20 
(2.1-2.9) 


2.6 + 0.20 
(2.1-3.0) 




Total Mortality Z 


- 


2.5 


12.8 


26.8 




Interval Undlssoclated 
Ammonia mg/1 NH3-N 


- 


0.22 + 0.036 
(0.17-0.27) 


0.20 + 0.037 
(0.16-0.27) 


0.20 + 0.040 
(0.14-0.27) 


3 


Mean Length ora 


2.5 + 0.22 

(2.cf::3.o) 


2.7 + 0.32 
(2.1-3.4) 


3.0 + 0.41 
(2.2-4.4) 


3.3 + 0.43 
(2.6^4.5) 




Total Mortality X 


- 


5 


7.5 


18.5 




Interval Undlssoclated 
Amnonla mg/1 NH3-N 


- 


0.12 ••- 0.018 
(0.10-0.14) 


0,10 + 0.026 
(0.07-0.14) 


0.10 + 0.023 
(0.07-0.14) 


2 


Mean Length mm 


2.5 + 0.26 
(1.9-3.1) 


3.1 + 0.45 
(2.3-4.1) 


3.5 + 0.53 
(2.7-4.8) 


3.9 + 0.62 
(2.9-5.2) 




Total Mortality X 


- 





2.5 


7.8 




Interval Undlssoclated 
Ammonia mg/1 NH3-N 


- 


0.01 + 0.002 
(0.004-0.01) 


0.01 + 0.002 
(0.004-0.01) 


0.01 + 0.002 
(0.003-0.01) 


1 
(control) 


Mean Length mm 


2.6 + 0.21 
(2.2-2.8) 


2.7 + 0.26 
(2.2-3.2) 


2.9 + 0.33 
(2.4-3.7) 


3.0 + 0.40 
(2.4-4.1) 




Total Mortality X 


- 


10 


18.9 


26.7 



•lean + standard deviation (Range) 



114 



APPENDIX B. PUBLICATIONS AND THESIS RESULTING FROM THIS RESEARCH. 



Publications 



Anderson, K.B., M.J. Sandusky, and R.E. Sparks. 1977. The toxicity of po- 
tassium, undissociated anunonia and Illinois River water to the fingernail 
clam ( Musculium transversum ) . Abstracts and program of the 39th Midwest 
Fish and Wildlife Conference: 35-36 (abstract). 

Anderson, K.B. , CM. Thompson, R.E. Sparks, and A. A. Paparo. 1976. Effects 
of potassium on adult Asiatic clams, Corbicula manilensis . Biological 
Notes No. 98. Illinois Natural History Survey. Urbana. 7 p. 

Anonymous. 1977. Of clams and ducks. The Illinois Natural History Survey 
Reports No. 164: 1-2. February. 

Murphy, J. A. and A. A. Paparo. 1976. Cytosomal and neuronal changes during 

photoreception as related to ionic permeability and ciliary activity ' 
in Mytilus edulis , p. 200-201. In: G.W. Bailey (ed.), 34th Ann. Proc. 
Electron Microscopy Soc. Amer. , Miami Beach, Florida. 

Paparo, A. A. 1976. The coordinate roles of branchial nerve activity and 
potassium in the stimulation of ciliary activity in Mytilus edulis : 
observations with phenoxybenz amine, bromolysergic acid and fluorescence 
histochemistry. J. Exp. Biol. 65: 109-116. 

Paparo, A. A. and J. A. Murphy. 1976. Light-dark changes in the morphology and 
elemental composition of pigment granules in the nerve ending under the 
lateral ciliated cell of the mussel, Mytilus edulis , p. 577-584. In: 
Scanning Electron Microscopy/ 1976 (Part VIII), Proceedings of the Workshop 
on Zoological Applications of SEM. IIT Research Institute. Chicago. 

Paparo, A.A. , K. Cunningham-Paparo, and J. Murphy. 1977. DOPA decarboxylase 
activities and potassium stimulation of lateral cilia on the gill of 
Mytilus edulis . I. A response to DOPA decarboxylase inhibitors and 
chemical sympathectomy. Bulletin of the Southern California Academy 
of Sciences 76(1): 32-37. 

Paparo, A.A. , K. Cunningham-Paparo, and J. Murphy. 1977. The effect of 

endogenous 5-HT on K ion enhancement of ciliary activity in the mussel 
Mytilus edulis . Bulletin of the Southern California Academy of Sciences 
76(2): 111-115. 

Paparo, A.A. and R.E. Sparks. 1977. Rapid assessment of water quality using 

the fingernail clam, Musculium transversum , p. 96-109. ^: J. Cairns, Jr., 
K.L. Dickson, and G.F. Westlake (eds.), Biological Monitoring of Water 
and Effluent Quality. American Society for Testing and Materials Special 
Publication 607. Philadelphia. 



115 



Paparo, A. A., R.E. Sparks, J. A. Murphy, and K.J. Cunningham-Paparo. 1977. 
The effect of potassium ions on the rate of ciliary activity in 
Sphaerium transversum . I. A different response in small and large 
clam preparations. Bulletin of the Southern California Academy of 
Sciences 76(3): 139-145. 

Sparks, R.E. and K.B. Anderson. 1977. Assessing toxicities in surface 

waters, with emphasis on the Illinois River. Abstracts with programs. 
North Central Section, Eleventh Annual Meeting, The Geological Society 
of America 9(5): 653 (abstract). 

Thompson, CM. and R.E. Sparks. 1977. Status of the fingernail clam 

( Musculium transversum ) in the Keokuk Pool, Mississippi River. Abstracts 
and program of the 39th Midwest Fish and Wildlife Conference: 24 
(abstract) . 



Thesis 

Anderson, K.B. 1977. Musculium transversum in the Illinois River and 
acute potassium bioassay method for the species. M.S. thesis. 
Western Illinois University. Macomb. 79 p.